EP0185756B1 - Extrusion techniques for producing liposomes - Google Patents
Extrusion techniques for producing liposomes Download PDFInfo
- Publication number
- EP0185756B1 EP0185756B1 EP85903513A EP85903513A EP0185756B1 EP 0185756 B1 EP0185756 B1 EP 0185756B1 EP 85903513 A EP85903513 A EP 85903513A EP 85903513 A EP85903513 A EP 85903513A EP 0185756 B1 EP0185756 B1 EP 0185756B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- liposomes
- filter
- filters
- pore size
- unilamellar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 229
- 238000000034 method Methods 0.000 title claims abstract description 159
- 238000001125 extrusion Methods 0.000 title abstract description 41
- 239000011148 porous material Substances 0.000 claims abstract description 80
- 150000002632 lipids Chemical class 0.000 claims abstract description 70
- 238000009826 distribution Methods 0.000 claims abstract description 63
- 239000000463 material Substances 0.000 claims abstract description 28
- 239000002904 solvent Substances 0.000 claims abstract description 27
- 239000003599 detergent Substances 0.000 claims abstract description 24
- 239000008188 pellet Substances 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims description 22
- 238000002296 dynamic light scattering Methods 0.000 claims description 12
- 239000012062 aqueous buffer Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 abstract description 12
- 150000003904 phospholipids Chemical class 0.000 description 27
- 239000004417 polycarbonate Substances 0.000 description 26
- 229920000515 polycarbonate Polymers 0.000 description 26
- 239000000523 sample Substances 0.000 description 25
- 238000004519 manufacturing process Methods 0.000 description 19
- 238000002360 preparation method Methods 0.000 description 19
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 17
- 229920001202 Inulin Polymers 0.000 description 16
- 229940029339 inulin Drugs 0.000 description 16
- 238000005311 autocorrelation function Methods 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 14
- 238000000527 sonication Methods 0.000 description 13
- 230000008569 process Effects 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 238000001000 micrograph Methods 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 9
- 238000005481 NMR spectroscopy Methods 0.000 description 9
- 239000002691 unilamellar liposome Substances 0.000 description 9
- 244000068988 Glycine max Species 0.000 description 8
- 235000010469 Glycine max Nutrition 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000013459 approach Methods 0.000 description 8
- 238000000502 dialysis Methods 0.000 description 8
- 238000001704 evaporation Methods 0.000 description 8
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 7
- 235000012000 cholesterol Nutrition 0.000 description 7
- 239000006185 dispersion Substances 0.000 description 7
- 230000008020 evaporation Effects 0.000 description 7
- 239000007789 gas Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 238000004513 sizing Methods 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- 229920005654 Sephadex Polymers 0.000 description 5
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000009792 diffusion process Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- -1 such as Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 230000007704 transition Effects 0.000 description 5
- 229960003732 tyramine Drugs 0.000 description 5
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical compound [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 description 5
- 238000004679 31P NMR spectroscopy Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- LFHISGNCFUNFFM-UHFFFAOYSA-N chloropicrin Chemical compound [O-][N+](=O)C(Cl)(Cl)Cl LFHISGNCFUNFFM-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 4
- 238000004064 recycling Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 239000002356 single layer Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003012 bilayer membrane Substances 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000000881 depressing effect Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 239000011800 void material Substances 0.000 description 3
- 238000003260 vortexing Methods 0.000 description 3
- CITHEXJVPOWHKC-UUWRZZSWSA-N 1,2-di-O-myristoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCC CITHEXJVPOWHKC-UUWRZZSWSA-N 0.000 description 2
- FJQZXCPWAGYPSD-UHFFFAOYSA-N 1,3,4,6-tetrachloro-3a,6a-diphenylimidazo[4,5-d]imidazole-2,5-dione Chemical compound ClN1C(=O)N(Cl)C2(C=3C=CC=CC=3)N(Cl)C(=O)N(Cl)C12C1=CC=CC=C1 FJQZXCPWAGYPSD-UHFFFAOYSA-N 0.000 description 2
- 101100000418 Autographa californica nuclear polyhedrosis virus Ac34 gene Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 229920005372 Plexiglas® Polymers 0.000 description 2
- 241000700157 Rattus norvegicus Species 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 239000003715 calcium chelating agent Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229960003724 dimyristoylphosphatidylcholine Drugs 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 230000000887 hydrating effect Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000003637 steroidlike Effects 0.000 description 2
- HVLLSGMXQDNUAL-UHFFFAOYSA-N triphenyl phosphite Chemical compound C=1C=CC=CC=1OP(OC=1C=CC=CC=1)OC1=CC=CC=C1 HVLLSGMXQDNUAL-UHFFFAOYSA-N 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 244000291564 Allium cepa Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010015719 Exsanguination Diseases 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 229910020889 NaBH3 Inorganic materials 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- AQLMHYSWFMLWBS-UHFFFAOYSA-N arsenite(1-) Chemical compound O[As](O)[O-] AQLMHYSWFMLWBS-UHFFFAOYSA-N 0.000 description 1
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- CPBQJMYROZQQJC-UHFFFAOYSA-N helium neon Chemical compound [He].[Ne] CPBQJMYROZQQJC-UHFFFAOYSA-N 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical group I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- ICIWUVCWSCSTAQ-UHFFFAOYSA-N iodic acid Chemical class OI(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-N 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical group 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000013022 venting Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
- B01D61/18—Apparatus therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
- B01D63/08—Flat membrane modules
Definitions
- This invention relates to liposomes and in particular to extrusion techniques for the rapid production of unilamellar liposomes and for the production of liposomes having defined size distributions.
- liposomes are closed vesicles having a lipid bilayer membrane surrounding an aqueous core.
- liposomes of the following three types have been produced: 1) miltilamellar vesicles (MLVs) wherein each vesicle includes multiple concentric bilayer membranes stacked one inside the other in an onion skin arrangement; 2) small unilamellar vesicles (SUVs) having only one bilayer membrane per vesicle and having diameters ranging up to about 50 nm; and 3) large unilamellar vesicles (LUVs), again having only one bilayer membrane per vesicle, but in this case having diameters greater than about 50 nm and typically on the order of 100 nm and above.
- MMVs miltilamellar vesicles
- SUVs small unilamellar vesicles
- LUVs large unilamellar vesicles
- liposomes which have been developed include stable plurilamellar vesicles (SPLVs), monophasic vesicles (MPVs), and steroidal liposomes. Descriptions of these vesicles and methods for preparing them can be found in copending and commonly assigned U.S. Patent No. 4,522,803; No. 4,588,578; No. 4,721,612 respectively.
- liposomes are as carriers for a variety of materials, such as, drugs, cosmetics, diagnostic reagents, bioactive compounds, and the like. Liposomes are also widely used as scientific models for naturally occurring biological membrane systems.
- the present invention relates to improved methods for the production of liposomes.
- the invention relates to: 1) an improved method for producing unilamellar liposomes of both the large and small types; and 2) an improved method for producing liposomes having defined size distributions.
- LUVs large unilamellar liposomes
- an aqueous buffer is introduced into a mixture of phospholipid and an organic solvent to produce "inverted micelles", i.e., droplets of water stabilized in the organic solvent by being surrounded by a phospholipid monolayer. Evaporation of the solvent causes the micelles to coalesce and form the desired liposomes.
- inverted micelles i.e., droplets of water stabilized in the organic solvent by being surrounded by a phospholipid monolayer.
- Evaporation of the solvent causes the micelles to coalesce and form the desired liposomes.
- lipid, detergent and an aqueous solution are mixed together and sonicated to form the desired vesicles. Separation techniques, such as, gel filtration, are then used to remove the detergent and thus produce the finished liposomes.
- lipid is dissolved in a solvent, e.g., pentane or diethyl ether, and the lipid- solvent solution is infused into an aqueous solution under conditions that cause the solvent to vaporize and thus produce the desired liposomes.
- a solvent e.g., pentane or diethyl ether
- LUVs Other techniques which have been used to produce LUVs include fusion techniques whereby a population of SUVs is treated so as to cause individual SUVs to fuse with each other to form LUVs.
- U.S. Patent 4,078,052 to P. Demetrios Papahadjopoulos describes a technique wherein calcium ions are used to fuse SUVs into cochleate cylinders, and the cylinders are then treated with a calcium chelating agent such as EDTA to form the desired LUVs. Rapid freezing of SUVs, followed by slow thawing, has also been used to produce LUVs by fusion. See, for example, U. Pick, Archives of Biochemistry and Biophysics, 212:186 (1981).
- each of the foregoing techniques can be used to produce liposomes, none of these techniques are totally satisfactory.
- each of the commonly used LUV techniques involves combining the components making up the liposome with a lipid solubilizing agent, i.e., either an organic solvent or a detergent.
- a lipid solubilizing agent i.e., either an organic solvent or a detergent.
- solvents and detergents can adversely effect many materials, such as enzymes, which one may want to encapsulate in liposomes, and thus these techniques cannot be used with these materials.
- the possible presence of these potentially toxic agents is undesirable.
- the fusion techniques include similar drawbacks.
- the calcium ion/calcium chelating agent technique like the solvent and detergent techniques, involves the use and subsequent removal of materials in addition to those actually making up the finished liposomes, in this case, the chelating agent and the added calcium ions.
- these materials represent possible sources of contamination, limit the usefulness of the technique, and make the technique more complicated.
- this technique requires that the composition of the liposomes include some phosphatidylserine.
- this technique suffers from the drawback that the specific trapping capacity of the lipsomes produced by the technique drops off sharply at phospholipid concentrations above about 20 mg/ml.
- the SUV techniques have similar problems. For example, high energy sonication can cause oxidation and degradation of phospholipids and may damage solute molecules which one wants to capture in the interior space of the liposomes. Also, when performed using a sonication probe, high energy sonication can cause probe erosion, and if one with bath sonication in combination with radioactive materials, can produce a potentially hazardous aerosol. Low energy sonication is slow, can be destructive to phospholipid molecules, and cannot be used to prepare large quantities of liposomes. Further, the sonication approach results in low trapping efficiencies.
- the infusion type SUV procedures suffer the same problems as the LUV infusion procedures.
- the high pressure French press technique has its own set of problems, including difficulties in making the technique repeatable, the need for post-preparation filtration to remove those MLVs which have not been converted to SUVs, the need for expensive and cumbersome equipment capable of withstanding the high pressures used, and contamination of the product by disintegration of components of the apparatus which occurs during processing of the liposomes. See, for example, Boswor t h, et al., Journal of Pharmaceutical Sciences, 71:806 (1982). Also, this technique can only produce small liposomes having a low trapping efficiency.
- Ching-hsien Huang in Biochemistry, 8:344 (1969), described a multi-step technique for producing a homogeneous population of small unilamellar liposomes (SUVs) which involved sonicating a lipid suspension in a buffer for 2) hours, centrifuging the resulting product at 105,OOOxg for 1 hour to remove undispersed lipid, filtering the supernatant through an extensively washed 0.1 pm Sartorius filter, subjecting the filtrate to molecular sieve chromatography on a Sepharose 4B column which had previously been saturated with the lipid suspension and washed and equilibrated with the buffer, and collecting the second fraction eluted from the column.
- SUVs small unilamellar liposomes
- Liposomes were prepared by mechanical agitation or by the French press techniuqe of Barenholz, et al., FEBS Lett., 99:210 (1979). Those produced by mechanical agitation were sized using filters having pore sizes of between 0.2 and 1.0 pm, with the liposomes being passed twice through the smallest filter and, in some cases, twice through each of the filters. For dialysis, filters having pore sizes between 0.05 and 3 ⁇ m were used.
- the invention in accordance with certain of its aspects, provides a method for producing a population of substantially unilamellar liposomes which involves repeated extrusions at moderate pressures of previously formed liposomes through a filter having a pore size below a critical upper limit, specifically, below approximately 100 nm.
- the invention provides a variety of advantages over previously known systems for producing unilamellar liposomes, including the following: 1) the ability to form unilamellar vesicles from a wide range of lipids; 2) the ability to use high lipid concentrations (e.g., on the order of 300 umol/ml) so as to easily achieve high trapping efficiencies; 3) the ability to provide reproducible and very rapid production of unilamellar vesicles, and, in particular, large unilamellar vesicles, through the use of high extrusion flow rates and automatic or semi-automatic recycling of the liposomes through the filter; 4) the ability to produce liposomes of a desired size by using a single pore size filter with minimum filter clogging problems; 5) the ability to avoid the use of organic solvents and detergents; and 6) the ability to provide an overall relatively gentle process.
- Filters having a pore size of about 100 nm are still used in accordance with this aspect of the invention, but with a reduced number of passes through the filter.
- the invention provides a method for producing liposomes directly from a lipid powder or pellet by simply combining the powder or pellet with an aqueous buffer and then applying sufficient pressure to the lipid/buffer mixture to repeatedly pass it through a filter. If the filter has a pore size less than about 100 nm, substantially unilamellar liposomes are produced. If the filter has a pore size significantly above 100 nm, e.g., on the order of 200 nm, multilamellar lipsomes are produced. Significantly, in either case, the liposomes are completely solvent free, in that, not even chloroform, as has been used in the past to produce MLVs, is required for liposome production in accordance with these aspects of the present invention.
- the invention provides populations of liposomes having essentially unimodal distributions about mean diameters which are greater than 50 nm.
- the invention provides a method for increasing the homogeneity of a population of liposomes by repeatedly passing the liposomes through one or more filters of a constant pore size until the size distribution of the population becomes essentially unimodal.
- FIGs 1A and 1 B are schematic diagrams of apparatus suitable for practicing the present invention.
- the liposome suspension is recycled through the filter by hand, while in Figure 1 B, the recycling has been partially automated.
- Figure 2 shows the 31p NMR signal intensity arising from egg phosphatidylcholine (EPC) multilamellar vesicles (in the presence of 5 mM MnCl 2 ) as a function of the number of extrusions through polycarbonate filters with 100 nm (circles) and 200 nm (squares) pore sizes.
- the lipid concentration in all cases was between 30 and 60 umol/ml.
- Figure 3 shows four freeze-fracture micrographs of vesicles prepared by repeated extrusion of multilamellar vesicles of varying lipid composition through polycarbonate filters: (a) soya phosphatidylcholine (PC) MLVs extruded through a 100 nm filter; (b) soya PC-soya PS (1:1) MLVs extruded through a 100 nm filter; (c) soya PE-soya PS (1:1) MLVs extruded through a 100 nm filter; (d) soya PC MLVs extruded through a filter with a 200 nm pore size.
- PC phosphatidylcholine
- the arrow in part (d) indicates a cross fracture revealing inner lamellae. All micrographs have the same magnification and the direction of shadowing is indicated by the arrowhead in the bottom right corner of each section. In each case, the extrusion procedure was repeated 10 times on lipid systems containing 40-70 umol/ml phospholipid.
- Figure 4 shows the size distribution of soya PC vesicles obtained after 10 extrusions through a polycarbonate filter with a 100 nm pore size.
- the vesicle diameters were measured from freeze fracture micrographs employing the technique of van Venetie et al., (1980) J. Micros., 118:401-408.
- the black and half-tone columns represent vesicles that did and did not undergo freeze-thaw cycling, respectively.
- FIG. 5 shows the calorimetric behaviour of hydrated dipalmitoylphosphatidylcholine (DPPC) in large multilamellar vesicles (MLVs) and in large unilamellar vesicles prepared by the extrusion technique of the present invention (LUVETs).
- the MLVs were formed by vortexing a dry lipid film in the bottom of a test tube in the presence of a NaCl buffer at 50°C, whereas the LUVETs were formed by repetitive extrusion (10 times) of the MLVs (50 mg lipid/ml) through 100 nm pore size polycarbonate filters at 50°C. Scan rates of 2.0°K/min were employed.
- Figure 6 shows the trapping efficiency as a function of lipid concentration for liposomes prepared in accordance with the present invention both with (open circles) and without (closed circles) freeze-thawing. 14 C-inulin was used as a trap marker.
- Figure 7 shows the 31p NMR signal intensity arising from egg phosphatidylcholine (EPC) multilamellar vesicles (in the presence of 5 mM MnC1 2 ) as a function of the number of extrusions through polycarbonate filters with 50 nm (open circles) and 30 nm (solid circles) pore sizes. The lipid concentration in all cases was 100 mg/ml.
- EPC egg phosphatidylcholine
- Figures 8 and 9 show freeze-fracture micrographs of the vesicles of Figure 7 processed through 50 nm and 30 nm filters, respectively.
- the upper portion of the figure ( Figures 8A and 9A) was prepared after one extrusion (x1) through two stacked polycarbonate filters, and the lower portion ( Figures 8B and 9B) after ten extrusions (x 10).
- Figure 10 shows the clearance of 125
- the LUVETs were prepared in accordance with the present invention and were injected into the tail vein of 150-175 g female Wistar rats at a dose level of 0.5 umol phospholipid in 100 ⁇ l HBS.
- Urine was collected in metabolic cages. Blood was withdrawn and the animals sacrificed at the indicated times and the total amount of 125 ITI in the blood calculated assuming 4.9 ml blood per 100 g rat. Results are expressed as percentages of the total 125
- injected ⁇ s.e. (n 4).
- Figure 11 shows the long term tissue distribution of the LUVETs of Figure 10.
- the symbols correspond to liver (circles); carcass (triangles) and spleen (squares). Results are expressed as percentages of total 125 ITI in vivo (total 125
- injected minus amount excreted) ⁇ s.e. (n 4).
- the present invention provides extrusion techniques for producing: 1) populations of substantially unilamellar liposomes; and 2) populations of liposomes having substantially unimodal distributions (hereinafter referred to as the "unilamellar” and “unimodal” aspects of the invention, respectively).
- the invention allows liposomes to be produced without the use of any solvents, detergents or other extraneous materials (hereinafter referred to as the "solvent free” aspects of the invention).
- the populations of substantially unilamellar liposomes are produced by subjecting previously formed liposomes to multiple extrusions at moderate pressures through a filter having a pore size of less than or equal to about 100 nm.
- the previously formed liposomes can have a variety of compositions and can be prepared by any of the techniques now known or subsequently developed for preparing liposomes.
- the previously formed liposomes can be formed by the conventional technique for preparing MLVs, that is, by depositing one or more selected lipids on the inside walls of a suitable vessel by dissolving the lipids in chloroform and then evaporating the chloroform, adding the aqueous solution to be encapsulated to the vessel, allowing the aqueous solution to hydrate the lipid, and swirling or vortexing the resulting lipid suspension to produce the desired liposomes.
- This technique employs the most gentle conditions and the simplest equipment and procedures known in the art for producing liposomes. Also, this technique specifically avoids the problems with sonication or the use of detergents, solvents (other than chloroform) or other extraneous materials, discussed above.
- the liposomes which are to be repeatedly extruded through the filter can be prepared by simply mixing a lipid powder or pellet and buffer together and then directly extruding that mixture through the filter. If the filter has a pore size of less than about 100 nm, this procedure produces unilamellar liposomes, while if the pore size is substantially greater than 100 nm, multilamellar liposomes are produced. In either case, the procedure eliminates the use of all solvents, including chloroform.
- the liposomes making up the population can have a variety of compositions and can be in the form of multilamellar, unilamellar, or other types of liposomes or, more generally, lipid-containing particles, now known or later developed.
- the lipid-containing particles can be in the form of steroidal liposomes, stable plurilamellar liposomes (SPLVs), monophasic vesicles (MPVs), or lipid matrix carriers (LMCs) of the types disclosed in copending and commonly assigned U.S. Patent No. 4,522,803; No. 4,588,578; No. 4,610,868 respectively.
- the mean diameter of the population will depend on the manner in which the liposomes are to be used. For example, as recognized by persons skilled in the art, for diagnostic applications, mean diameters in the range of 100 nm to 500 nm are generally preferred, while for depoting of drugs, larger diameters, e.g., on the order of 500 nm to 1000 nm, are preferred, and for applications where endocytosis is desirable, smaller diameters, e.g., on the order of 50 nm to 100 nm, are preferred. Similar ranges have been recognized in the art for other applications. See, for example, Liposomes, supra.
- Mean diameters of populations of liposomes can be measured by various techniques known in the art, including freeze-fracture and quasi-elastic light scattering. As discussed above and in more detail below, quasi-elastic light scattering is preferred in the context of the unimodal aspects of the present invention, and the values for liposome diameters reported herein in connection with those aspects were measured using this technique.
- the substantially unimodal population of liposomes is prepared by repeatedly passing previously formed liposomes through filters of a constant pore size until the size distribution of the population in fact becomes unimodal. It has been surprisingly found that repeated passages through filters of a constant pore size causes bimodal aspects of the original size distribution of the liposomes, as well as any higher modal aspects, to eventually disappear. For the method to work, however, it is necessary to use one pore size, and use it repeatedly.
- the previously formed liposomes can be prepared by any of the techniques now known or subsequently developed for preparing liposomes.
- the previously formed liposomes can be formed by the conventional technique, discussed above, for preparing multilamellar liposomes (MLVs).
- LUVs large unilamellar liposomes
- reverse-phase evaporation infusion procedures, and detergent dilution
- detergent dilution a review of these and other methods for producing liposomes can be found in the text Liposomes, supra, the pertinent portions of which are incorporated herein by reference.
- the previously formed liposomes can be produced in accordance with the procedures described in U.S. Patent No. 4,522,803; No. 4,588,578; No. 4,721,612.
- other lipid-containing particles such as those described in U.S. Patent No.
- the resulting unimodal population will, in general, not be a population of liposomes, but rather a population having similar characteristics to those of the original lipid-containing particles used.
- the substantially unimodal population of liposomes can be prepared using the solvent free approach, discussed above. That is, the population of liposomes can be prepared directly from a lipid powder or pellet and buffer by simply mixing these ingredients together and then directly passing that mixture through filters of a selected pore size a sufficient number of times to achieve the desired unimodality.
- the filter used to generate the unilamellar liposomes or the unimodal distribution of liposomes is preferably of the type which has straight through channels.
- Polycarbonate filters of this type produced by Nuclepore, Inc., Pleasanton, CA, have been found to work successfully in the practice of the present invention.
- the filter may have to be changed after the first two or three passes of the liposome suspension due to pore clogging. Clogging in general depends on such variables as lipid composition, purity and concentration, as well as on the pressure and flow rates used.
- the most critical parameter in preparing unilamellar liposomes in accordance with the present invention is the size of the filter's channels. It has been found that unilamellar liposomes cannot be produced from multilamellar liposomes, no matter how many times the MLVs are passed through the filter, if the filter's pore size is significantly above about 100 nm, e.g., if the pore size is about 200 nm (see Example 2, infra). Accordingly, only filters having a pore size equal to or below about 100 nm can be used in connection with this aspect of the invention.
- the size of the unilamellar liposomes produced depends on the pore size of the filter used, the mean diameter being, in general, somewhat smaller than the pore size. If desired, the liposome's mean diameter, as well as their trapped volume (ul per umol phospholipid), can be easily increased using the freeze-thaw procedure discussed above (see also Example 4, infra). Importantly, since this procedure does not involve the use of solvents, detergents or other extraneous materials, the increase in liposome size is not at the expense of introducing contamination and degradation problems. Vesicle size and trapped volumes can also be manipulated by varying other parameters of the system, such as, lipid composition.
- the number of passes through the filter needed to produce the desired unilamellar liposomes depends on the filter characteristics (pore size, composition and geometry) and the materials from which the liposomes are to be made. As illustrated by Example 2, infra, five or more passes through a double stacked polycarbonate filter having a pore size of 100 nm are typically required to obtain unilamellar liposomes. Less passes may be needed for smaller pore sizes. For example, with 30 nm and 50 nm filters, two to four passes are in general sufficient to produce a substantially unilamellar population of liposomes (see Example 6, infra).
- the pore size of the filter is the primary determinant of the mean diameter of the final population. In general, within approximately 15 to 50 percent, the mean diameter is approximately equal to the pore size. However, for pore sizes below about 100 nm, the mean diameter of the population tends to level off at about 75 nm as measured by quasi-elastic light scattering, irrespective of the specific pore size used. As discussed above, for pore sizes below about 100 nm, the finished liposomes are found to be substantially unilamellar, irrespective of the lamellarity of the original population. For pore sizes above 100 nm, multilamellar liposomes remain multilamellar and unilamellar liposomes remain unilamellar.
- the number of passes through the filter needed to produce a substantially unimodal population depends on the filter characteristics (pore size, composition and geometry) and the materials from which the liposomes are to be made. In some cases three to five passes through double stacked polycarbonate filters are sufficient to produce a unimodal population. As a general proposition, 25 passes through double stacked polycarbonate filters will produce the desired unimodal distribution for most liposome preparations. The appropriate number of passes for any particular system can easily be determined by persons of ordinary skill in the art by simply sampling the finished liposomes to determine when substantial unimodality has been achieved.
- Passage of the liposomes through the filter to produce the unimodal, unilamellar, or both unimodal and unilamellar population of liposomes is accomplished under pressure. Pressures of various magnitudes can be used depending upon the type and composition of the liposomes to be produced, the specific characteristics of the equipment employed, and the rate at which liposomes are to be produced.
- the appropriate pressure for a particular system can be readily determined by persons skilled in the art by examining the finished liposomes to determine if they are substantially intact and have the desired unimodal distribution and/or unilamellarity.
- Pressures in the 100-700 psi (689.47-4,826.29 kPa) range are also preferred because they allow for the extrusion of solutions having lipid concentrations on the order of about 300 umols phospholipid per ml without significant filter clogging.
- Prior art liposome sizing techniques employing polycarbonate filters used pressures less than 100 psi (689.47 kPa), and thus were limited to lipid concentrations of 60 umol/ml.
- the use of high lipid concentrations has resulted in trapping efficiencies on the order of 30% for the present invention. Rapid extrusion rates on the order of 20 ml/min and above are still achieved for such high lipid concentrations when pressures in the range of 300-500 psi (2,068.41-3,447.35 kPa) are used.
- Flow rates on the order of 20-60 ml/min do not represent the maximum flow rates that are achievable with the present invention, but merely represent rates consistent with convenient collection of the extruded material.
- Maximum flow rates are sensitive to the concentration of lipid, the history of the sample (i.e., whether it has been extruded one or more times), the pressure employed, and the nature of the lipids themselves. For example, "gel state" lipids cannot be extruded. Such lipids (e.g., dipalmitoyl phosphatidylcholine (DPPPC)) must be heated above their gel to liquid crystalline transition temperature (41°C for DPPC) before the extrusion process can proceed.
- DPPPC dipalmitoyl phosphatidylcholine
- the extrusion can be exceedingly rapid.
- a 5 ml dispersion of 50 mg/ml egg phosphatidylcholine (EPC) has been extruded through a 100 nm pore size filter in less than 2 seconds at 500 psi (3,447.35 kPa), corresponding to a flow rate of at least 150 ml/min. This rate would be increased further by higher pressure or temperature.
- EPC egg phosphatidylcholine
- FIG. 1A Suitable apparatus for practicing the present invention is shown in Figures 1A and 1B.
- liposome suspension 4 which is to be extruded through filter 6 is introduced into pressure chamber 2 by means of injection port 8.
- the injection port also serves as a release valve.
- Pressure chamber 2 is formed from upper portion 10 and lower portion 12 which are connected together by, for example, bolts 14.
- a seal between these portions and filter 6 is provided by O-ring 16.
- the chamber is made of clear plastic so that the extrusion of the suspension from the chamber can be visually observed.
- the filter is supported within chamber 2 by filter support 18. In practice, it has been found convenient to use two stacked polycarbonate filters to form filter 6.
- Pressure is supplied to chamber 2 by means of conduit 20 which is connected to a source of pressure, e.g., a high pressure nitrogen tank (not shown).
- Conduit 20 includes valve and regulator 22 for adjusting the pressure within chamber 2.
- the material extruded from filter 6 is removed from chamber 2 by means of conduit 24 and collected in receiving vessel 26.
- valve 22 is closed prior to the time all of the suspension has been extruded from chamber 2 so as to prevent high pressure gas from flowing through the system and blowing the suspension out of receiving vessel 26.
- the extruded material is repeatedly returned to chamber 2 by means of injection port 8, until the original population of liposomes has passed through filter 6 a sufficient number of times so as to substantially increase its unimodality and/or unilamellarity.
- Figure 1B shows a modification of the apparatus of Figure 1A wherein the recycling of the extrudate is partially automated.
- the apparatus shown in this figure is used as follows.
- a filter 6 is installed in the apparatus by removing threaded retainer plug 5, filter support housing 9, and 0-ring 16 from aluminum housing 7.
- a filter is then placed on the filter support and the components reassembled with plug 5 being tightened until 0-ring 16 is compressed against inner plexiglas housing 11 contained within outer aluminum housing 13.
- a porous drain disc (not shown) can be placed under the filter.
- a sample is then loaded into receiving chamber 3 by rotating load/recycle/discharge valve 15 until load/ discharge tube 17 is aligned with inlet port 19, and by rotating pressure/vent valve 23 until vent port 25 is aligned with exhaust port 21.
- a sample can then be introduced into the receiving chamber through load/ discharge tube 17. Most conveniently, this is done by attaching a short length of flexible tubing and a hypodermic syringe to load/discharge tube 17.
- Pressure is then applied to pressure chamber 2 causing the sample to pass from that chamber to receiving chamber 3 by way of filter 6, bypass port 29, recycle port 37, and inlet port 19. This accomplishes one extrusion of the sample through the filter. Flow from pressure chamber 2 to receiving chamber 3 can be visually observed through plexiglas housing 11, and the amount of pressure applied can be adjusted to achieve a gentle flow.
- the sample is discharged from the apparatus through load/discharge tube 17 by first aligning gas inlet port 31 with exhaust port 21 and load/discharge tube 17 with inlet port 19, and then applying pressure to the system from the external pressure source.
- the external pressure is shut off before all of the sample has left chamber 3 to avoid high gas flows at the end of the evacuation.
- the sample can also be removed by disassembling threaded retainer plug 5, filter support housing 9, and 0-ring 16 from aluminum housing 7, and then depressing transfer valve 27 to cause the sample to flow into the pressure chamber and out the bottom of the apparatus.
- the apparatus shown in Figure 1B, or equivalent apparatus can be equipped with conventional automatic fluid handling equipment and controls to achieve a completely automated process. Also, so as to be able to maintain the temperature of the sample above the gel to liquid crystalline transition temperature of the lipids used, the pressure chambers of whatever equipment is employed can be heated using a water jacket or similar device.
- EPC Egg phosphatidylcholine
- SPC soya phosphatidylcholine
- PE phosphatidylethanolamine
- PS phosphatidylserine
- the lipids from the soya source are considerably more unsaturated than those derived from EPC, due to the high content of linoleic acid in SPC (see Tilcock, C. P. S. and Cullis, P. R. (1981) Biochim. Biophys. Acta, 641:189-201). All lipids were more than 95% pure as determined by TLC. Acidic phospholipids (PS) were converted to the sodium salt form as described in Hope, M. J. and Cullis, P. R. (1980) Biochim. Biophs. Res. Commun., 92:846-852. Cholesterol (Sigma, St. Louis) was used without further purification.
- multilamellar vesicles were prepared in accordance with the procedures described in Example 1, infra, but in the presence of 1 uCi of 22 Na or 14 C-inulin (NEN, Canada). Unilamellar liposomes were then prepared from the multilamellar liposomes, again following the procedures of Example 1.
- 31 P NMR was employed to provide an indication of the extent to which the vesicle preparations were unilamellar. Specifically, Mn 2+ was added to the vesicle dispersion (3 ml, 30-60 umol phospholipid per ml in a 15 mm diameter NMR tube) at levels (5 mM) sufficient to broaden beyond detection the 31 P NMR signal from those phospholipids facing the external medium. If the vesicles are large and unilamellar, then approximately 50% of the signal should remain following the addition of Mn 2+ . The impermeability of the vesicles to Mn 2+ was demonstrated by following the timecourse of the signal intensity, which for the PC systems investigated was found to be stable over a period of days.
- Spectra were obtained employing a Bruker WP 200 NMR spectrometer operating at 81 MHz. Accumulated free induction decays corresponding to 1000 transients were collected using a 15 usec 90° radiofrequency pulse, gated proton decoupling and a 20 KHz sweep width. An exponential multiplication corresponding to a 50 Hz linebroadening was applied prior to Fourier transformation. Signal intensities were measured by cutting out and weighing spectra with triphenylphosphite (in a small central capillary in the NMR tube) as a reference.
- Vesicle size distributions for Examples 1-7 and part of Example 8 were determined by freeze-fracture. Vesicle preparations were mixed with glycerol (25% by volume) and frozen in a freon slush. Samples were fractured and replicated employing a Balzers BAF 400D apparatus, and micrographs of replicas were obtained using a Phillips 400 electron microscope. Vesicle size distributions were determined by measuring the diameter of fractured vesicles that were 50% shadowed according to the procedure of van Venetie et a/., (1980) J. Micros., 118:401-408.
- Vesicle size distributions for part of Example 8 and Examples 9-10 were determined using quasi-elastic light scattering, also known as dynamic light scattering or photon-correlation spectroscopy.
- this technique involves passing coherent light, e.g., light produced by a helium- neon laser, through a sample of the suspended vesicles and measuring the time dependent fluctuations in the intensity of the light scattered by the vesicles.
- An autocorrelation function is then calculated from this data.
- this autocorrelation function is directly related to the diffusion coefficients of the vesicles in the sample, which in turn are a function of the hydrodynamic radii of the vesicles. Accordingly, different vesicle size distributions will produce different autocorrelation functions.
- the extent to which a second order polynomial, i.e., a quadratic, in fact does fit the natural logarithm of the autocorrelation function for a particular sample is an accurate measure of the extent to which the diffusion coefficients of the vesicles in the sample have a unimodal Gaussian distribution.
- this procedure is often referred to as a cumulants analysis of the autocorrelation function.
- a population of liposomes is considered to be substantially "unimodal" when the logarithm of its autocorrelation function fits closely to a second order polynomial.
- autocorrelation functions were obtained using a Nicomp Model 200 Laser Particle Sizer (Nicomp Instruments, Inc., Santa Barbara, California). This equipment uses the standard method of least-squares for curve-fitting and reports goodness of fit as a chi 2 value derived from the deviations between the logarithm of the autocorrelation function and the values predicted by the second order polynomial. Values of chi 2 in the range of 0-2 indicate a good fit of the data by the assumed unimodal Gaussian distribution, while high values of chi 2 indicate a poor fit.
- an estimate of the standard deviation of the distribution can be derived from the square root of the coefficient of the second order term of the polynomial. For distributions having a good fit, this estimate is reported in the examples, while for poor fits, the estimate is not reported, since, although a value can be calculated, such a value does not in fact serve as an estimate of the standard deviation.
- liposomes prepared in accordance with this technique are referred to herein by the acryonym "LUVETs", i.e., Large U nilamellar Vesicles by Extrusion Techniques.
- LVETs Large U nilamellar Vesicles by Extrusion Techniques.
- MLVs large multilamellar vesicles
- lipid dissolved in chloroform was dried down and deposited as a film on the inside of a test tube.
- the MLVs were then formed by simply adding an aqueous buffer of 150 mM NaCl, 20 mM HEPES, pH 7.5, to the test tube and hydrating the lipid by vortex mixing.
- the resulting MLV dispersion (2-10 ml) was then transferred into the pressure chamber of the apparatus shown in Figure 1A, equipped with two stacked standard 25 mm polycarbonate filters having a 100 nm pore size (Nuclepore, Inc., Pleasanton, California, Catalog # 110605). Nitrogen pressure was applied to the chamber via a standard gas cylinder fitted with a high pressure (0-4000 psi (2,757.88 kPa)) regulator. The vesicles were extruded through the filter employing pressures of 100-700 psi (689.47-4,826.29 kPa) resulting in flow rates of 20-60 ml/min, and were collected and re-injected. Repetition of the extrusion procedure five or more times resulted in the production of large unilamellar liposomes having diameters of approximately 70 nm as measured by freeze fracture. The overall extrusion process including recycling generally took fifteen minutes or less.
- This example demonstrates the criticality of filter pore size in producing unilamellar liposomes, and, in particular, the criticality of using a filter having a pore size of less than or equal to about 100 nm.
- EPC MLVs were prepared in accordance with the procedures described in Example 1 and then repeatedly passed through polycarbonate filters having pore sizes of 100 and 200 nm.
- the unilamellarity of the resulting liposomes was determined using the 31p NMR technique described above. The results are shown in Figure 2.
- the signal intensity drops to approximately 65% after five passes through the filter and then remains relatively constant.
- the signal drops to approximately 50% after five or more passes.
- the half-tone columns in Figure 4 show the vesicle diameters measured for SPC LUVETs which were prepared by passing MLVs prepared in accordance with Example 1 through two (stacked) 100 nm pore size filters ten times.
- Table I shows in summary form the measured mean diameters and mean trapped volumes for this and other lipid compositions.
- EPC LUVs were prepared by two procedures (octylglucoside detergent dialysis and reverse phase evaporation) which are generally accepted to produce unilamellar vesicles, and the LUVs so produced were then extruded ten times through two (stacked) 100 nm pore size filters. See Mimms, L.
- the vesicle diameter distribution shown in Figure 4 can be used to determine calculated values for trapped volumes and amount of inner monolayer phospholipid by assuming 1) an area per phospholipid molecule, e.g., 0.6 nm 2 (see Schieren, H., Rudolph, S., Finkelstein, M., Coleman, P. and Weissman, G. (1978) Biochim. Biophys. Acta., 542:137-153); 2) a bilayer thickness, e.g., 4 nm (see Blaurock, A. E. (1982) Biochim. Biophys. Acta., 650:167-207); and 3) that the vesicles are unilamellar. These calculated values can then be compared with the experimentally observed trapped volumes and amounts of inner monolayer phospholipid to determine the proportion of unilamellar vesicles present.
- the trapped volumes measured for EPC LUVs produced by the octylglucoside detergent dialysis procedure and the reverse phase evaporation procedure, which were subsequently extruded 10 times through a filter with a 100 nm pore size are comparable to the trapped volumes obtained for the EPC LUVETs.
- SUVs composed of saturated phospholipids, such as, DPPC are known to exhibit a reduction in the gel-liquid crystalline transition temperature (T e ) and a broadening of the transition due to their highly curved membranes. This high curvature is generally considered undesirable because it results in increased disorder in the membrane's hydrocarbon region (see Schuh, J. R. Banerjee, U., Muller, L. and Chan. S. I. (1982) Biochim. Biophys. Acta, 687:219-225).
- T e values were calorimetrically measured for MLVs and LUVETs prepared in accordance with the procedures of Example 1. The results are shown in Figure 5.
- the MLVs and LUVETs exhibit very similar values of T c . These values are consistent with those reported in the literature. See Ladbrooke, B. D. and Chapman, D. (1969) Chem. Phys. Lipids, 3:304-367. They are in direct contrast to the behavior observed for sonicated DPPC vesicles, which exhibit a broadened gel-liquid crystalline transition which occurs some 4°C below the melting temperature of the multilamellar systems. See van Dijck, P. W. M., de Kruijff, B., Aarts, P. A. M. M., Verkleij, A. J. and de Gier, J. 1978) Biochim. Biophys. Acta, 506:183-191. Accordingly, the unilamellar liposomes prepared by the procedures of the present invention using filters with a 100 nm pore size are properly classified as LUVs, rather than SUVs.
- LUVETs were prepared in accordance with the procedures of Example 1, but with a buffer having a NaCl concentration of 1 M, instead of 150 mM. After preparation, the liposomes were placed in distilled water creating a large osmotic pressure difference across the liposomes' membranes. Using arenazo III as a marker for liposome leakage, essentially no leakage was found under these severe test conditions.
- This example illustrates the use of a freeze-thaw procedure to increase the trapped volumes of the unilamellar liposomes produced by the present invention.
- SPC and EPC LUVETs prepared in accordance with the procedures of Example 1 were subjected to two freeze-thaw cycles (employing liquid nitrogen) followed by extrusion through new 100 nm pore size filters. Specifically, the LUVETs were placed in a plastic vial, and the vial placed in liquid nitrogen for approximately 1 minute. The frozen LUVETs were then thawed in water at room temperature for approximately 5 minutes. The thawed solution was extruded 3 times through new 100 nm filters, after which the freeze-thaw-extrude process was repeated a second time.
- the mean diameter of the SPC LUVETs increased by approximately 20 nm.
- the calculated trapped volume for this vesicle distribution is 2.3 ul/umole which is in excellent agreement with the measured value of 2.2 ⁇ 0.1 ul/umol (Table I).
- LUV preparations An important parameter of LUV preparations is their trapping efficiency. This is especially so when the agents to be trapped are either expensive, as is the case for many drugs, or have low solubilities.
- This example illustrates the effects of using filters having pore sizes less than 100 nm on the size of the liposomes produced and on the number of passes through the filter needed to achieve substantial unilamellarity.
- MLVs were prepared in accordance with the procedures of Example 1 using egg PC at a concentration of 100 mg/ml and using a buffer of 150 mM NaCI and 20 mM Hepes (pH 7.5). The dispersion was passed ten times through two stacked polycarbonate filters having a pore size of either 50 nm or 30 nm using the apparatus of Figure 1A. Aliquots were taken after one and afterten passes through the extrusion device and used to prepare freeze-fracture micrographs as described above. Samples (4 ml, 25 mg phospholipid per ml) were also taken after various numbers of passes and analyzed by 31p NMR using Mn 2+ as described above. The results are shown in Figures 7-9.
- This example illustrates the use of liposomes prepared in accordance with the present invention to deliver entrapped material in vivo.
- it illustrates for a rat model system the administration and subsequent in vivo distribution of 121
- Tyraminyl-inulin was prepared as follows. Inulin (1.0 g) was dissolved in 90.0 ml distilled H 2 0 and cooled to 4°C, 10 ml (fresh) 0.1 M peridic acid was added and the solution was incubated at 4°C for 15 minutes in the dark. Periodate consumption was assayed by the arsenite method indicating approximately two oxidations per inulin molecule (see Dyer, J. in Methods of Biochemical Analysis, P. Glick (Ed.), Vol. 3, p. 111, Interscience (1956)). The reaction was terminated by neutralization with Ba(OH) 2 and the periodate and iodate salts were removed by centrifugation.
- the fractions were assayed for tyramine by monitoring the absorbance at 279 nm and for sugar by employing the anthrone reagent technique (see Roe, J. H. (1955) J. Bio. Chem., 212:335-343).
- the sugar containing fraction eluted in the void volume and had a constant tyramine:inulin mole ratio of 0.6.
- the adduct was completely separated from the free tyramine and other salts as determined by rechromatography on Sephadex @ G-25. The peak fractions were lyophilized giving an 80% yield, based on inulin.
- the tyraminyl-inulin adduct was iodinated as follows. 2.5 mg of the tyraminyl-inulin adduct were dissolved in 0.2 ml HEPES (20 mM), NaCl (145 mM) pH 7.4 (HEPES buffered saline; HBS) and placed in a 1.5 ml stoppered vial in which 40 ug iodogen had been previously deposited from 300 uL CHCI 3 . Then carrier free Na1251 (4 mCi, 100 mCi/ml) was added and the reaction allowed to proceede for 45 min at room temperature.
- -tyraminyl-inulin ( 125 lTl) solution routinely contained 1 uCi/uL 125 1, where less than 0.01 % was in the free iodide form (less than 0.01 % was CHCI 3 extractable when made to 1.2% H 2 0 2 and 0.4% KI) and over 99% of the material eluted as one peak in the void volume on re-chromatography employing Sephadex G-25. In all studies the material was used within 2 weeks of production.
- were prepared in accordance with the procedures described above. Specifically, 30 umol egg phosphatidylcholine (EPC) and 30 umol cholesterol were dried down from CHCI 3 . The resulting lipid film was dispersed in 1 ml HBS containing 1 mCi 125
- the LUVET's were administered to female Wistar rats (150-200 g), which were fed ad libitum prior to and during the experiments, by lightly anesthetizing the animals with ether and then injecting 200 ul HBS containing approximately 0.5 uCi 125
- the rats were allowed to recover in metabolic cages where the urine and feces were collected. At various times post injection the rats were anesthetized with ether and sacrificed by exsanguination via the vena cava.
- Blood was collected in a syringe containing 200 uL 200 mM EDTA and recovery was approximately 85% assuming 4.9 ml blood/100 g rat.
- the heart, liver, lung, spleen and kidney were removed and the urine remaining in the bladder was collected.
- the carcass was then dissolved in 200 ml 9 M NaOH at 70°C overnight. Aliquots of carcass digest and samples of tissues were then assayed for the presence of 125
- Figure 10 illustrates the clearance from the circulation of the LUVETs and the subsequent appearance of inulin in the urine.
- the encapsulated material in the circulation is initially rapidly reduced to approximately 40% of the injected levels, and thereafter decays with a much longer half-life (approximately 3 hr). Further, only 30% of the injected dose is eventually found in the urine even after 3 days. This latter result clearly indicates tissue uptake and retention of LUVET encapsulated 125
- This example illustrates the production of liposomes directly from a lipid powder or pellet and buffer without the use of any solvents or other extraneous materials.
- Freeze fracutre micrographs of the resulting product were prepared following the procedures described above.
- the product was found to be a homogeneous population of substantially unilamellar liposomes having a mean diameter of approximately 70 nm as measured by freeze fracture. If desired, this mean diameter can be increased using the freeze-thaw procedures of Example 4 above.
- This example illustrates the preparation of a population of liposomes having a substantially unimodal distribution.
- MLVs Large multilamellar vesicles
- EPC prepared as described above was dissolved in chloroform and dried down and deposited as a film on the inside of a test tube.
- the MLVs were then formed by simply adding an aqueous buffer of 150 mM NaCI, 20 mM HEPES, pH 7.5, to the test tube and hydrating the lipid by vortex mixing.
- the resulting MLV dispersion was then transferred into the pressure chamber of the apparatus shown in Figure 1A, equipped with two stacked standard 25 mm polycarbonate filters having a pore size of 200 nm (Nuclepore, Inc., Pleasanton, California, Catalog # 110606).
- the dispersion was extruded through the filters 25 times at a temperature of 20°C.
- the pressures employed were on the order of 100 psi (689.47 kPa), and the resulting flow rates were on the order of 30 ml/min.
- the sizing procedure was completed in less than approximately 15 minutes, and the resulting liposomes were found to be substantially intact, nothwithstanding their many passes through the filters.
- the size distribution of the population at the end of the 25 passes was determined using the quasi-elastic light scattering technique described above. The results are shown in Table II, infra.
- the population had a chi 2 value of 1.42 indicating that a good fit was achieved by a second order polynomial, and thus that the diffusion coefficients of the vesicles had a unimodal Gaussian distribution.
- the mean diameter calculated for this population is 168 nm, i.e., about 15% smaller than the 200 nm pore size used for extrusion, and the standard deviation about the mean is a relatively narrow 55 nm.
- This example demonstrates the importance of using filters of a constant pore size, as opposed to a sequence of filters of decreasing pore sizes, to obtain a population of liposomes having a substantially unimodal size distribution.
- MLVs were prepared as in Example 9, but instead of being extruded 25 times through filters having the same pore size, they were extruded once through a series of filters having the following pore sizes: 1000 nm, 800 nm, 600 nm, 400 nm and 200 nm (Nuclepore, Inc., Pleasanton, California, Catalog Nos. 110610, 110609, 110608, 110607, and 110606).
- the apparatus of Figure 1A was used, equipped in this case with just a single filter for each filter size.
- the pressures, flow rates and processing temperature were the same as in Example 9.
- the size distribution of the liposomes prepared in this manner, as determined by quasi-elastic light scattering, is shown in Table II.
- a huge chi 2 value of 368 was calculated, which means that a second order polynomial did not fit the data, and thus that the diffusion coefficients of the liposomes do not have a unimodal Gaussian distribution.
- Example 9 Comparing this result with the results for Example 9 clearly establishes that multiple passes of liposomes through filters of a constant pore size surprisingly produce a materially different size distribution from that produced by passage of the same type of liposomes through a series of filters of decreasing pore size.
Abstract
Description
- This invention relates to liposomes and in particular to extrusion techniques for the rapid production of unilamellar liposomes and for the production of liposomes having defined size distributions.
- As is well known in the art, liposomes are closed vesicles having a lipid bilayer membrane surrounding an aqueous core. In general, liposomes of the following three types have been produced: 1) miltilamellar vesicles (MLVs) wherein each vesicle includes multiple concentric bilayer membranes stacked one inside the other in an onion skin arrangement; 2) small unilamellar vesicles (SUVs) having only one bilayer membrane per vesicle and having diameters ranging up to about 50 nm; and 3) large unilamellar vesicles (LUVs), again having only one bilayer membrane per vesicle, but in this case having diameters greater than about 50 nm and typically on the order of 100 nm and above.
- A review of these three types of liposomes, including methods for their preparation and various uses for the finished liposomes, can be found in the text Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, see also Szoka, Jr., et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980).
- Other types of liposomes which have been developed include stable plurilamellar vesicles (SPLVs), monophasic vesicles (MPVs), and steroidal liposomes. Descriptions of these vesicles and methods for preparing them can be found in copending and commonly assigned U.S. Patent No. 4,522,803; No. 4,588,578; No. 4,721,612 respectively.
- One of the primary uses for liposomes is as carriers for a variety of materials, such as, drugs, cosmetics, diagnostic reagents, bioactive compounds, and the like. Liposomes are also widely used as scientific models for naturally occurring biological membrane systems.
- In connection with each of these uses, it is important to have available populations of liposomes which have defined mean diameters and defined size distributions about those means. More particularly, it is important to have available populations of liposomes which have a substantially unimodal distribution about a selected mean diameter.
- In terms of commercial applications, and in particular, pharmaceutical applications, such populations are needed to enhance the effectiveness and safety of liposome encapsulated drugs and similar materials. Moreover, the availability of accurately defined populations of liposomes would make it significantly easier to obtain approval for liposome-containing preparations from such regulatory agencies as the United States Food and Drug Administration. In terms of other liposome applications, including scientific investigations, the ready availability of well-characterized populations of liposomes would lead to more standardized products and repeatable experiments.
- The present invention relates to improved methods for the production of liposomes. In particular, the invention relates to: 1) an improved method for producing unilamellar liposomes of both the large and small types; and 2) an improved method for producing liposomes having defined size distributions.
- Prior to the present invention, large unilamellar liposomes (LUVs) were commonly produced by one of the following three methods; 1) reverse-phase evaporation, 2) detergent dilution, and 3) infusion procedures using various solvents. See Liposomes, supra, Ch. 1, pages 37-44.
- In the reverse-phase evaporation technique, an aqueous buffer is introduced into a mixture of phospholipid and an organic solvent to produce "inverted micelles", i.e., droplets of water stabilized in the organic solvent by being surrounded by a phospholipid monolayer. Evaporation of the solvent causes the micelles to coalesce and form the desired liposomes. See, for example, Szoka, Jr., et al., Proc. Natl. Acad. Sci. USA, 75:4194 (1978); and U.S. Patent 4,235,871 to Papahadjopoulos et al.
- In the detergent dilution approach, lipid, detergent and an aqueous solution are mixed together and sonicated to form the desired vesicles. Separation techniques, such as, gel filtration, are then used to remove the detergent and thus produce the finished liposomes.
- In the infusion procedures, lipid is dissolved in a solvent, e.g., pentane or diethyl ether, and the lipid- solvent solution is infused into an aqueous solution under conditions that cause the solvent to vaporize and thus produce the desired liposomes. See, for example, Deamer, Annals New York Academy of Sciences, 308:250-258 (1978).
- Other techniques which have been used to produce LUVs include fusion techniques whereby a population of SUVs is treated so as to cause individual SUVs to fuse with each other to form LUVs. For example, U.S. Patent 4,078,052 to P. Demetrios Papahadjopoulos describes a technique wherein calcium ions are used to fuse SUVs into cochleate cylinders, and the cylinders are then treated with a calcium chelating agent such as EDTA to form the desired LUVs. Rapid freezing of SUVs, followed by slow thawing, has also been used to produce LUVs by fusion. See, for example, U. Pick, Archives of Biochemistry and Biophysics, 212:186 (1981).
- With regard to the production of SUVs, as with LUVs, a variety of techniques have been employed in the past. See Liposomes, supra, Ch. 1,
pages 33, 36. The earliest technique involved sonication to clarity of a suspension of lipid in an aqueous solution using a probe or bath sonication unit. Other techniques have included infusion procedures along the lines of those used for producing LUVs but with ethanol as the solvent (see S. Batzri and E. Korn, Biochimica et Biophysica Acta, 298:1015 (1973)), and a technique employing multiple passes of MLVs through a French press operated at a pressure of 20,000 psi (13,7894 kPa) (see, for example, Hamilton, Jr., et al., Journal of Lipid Research, 21:981 (1980); and Barenholz, et al., FEBS Lett., 99:210 (1979)). - In addition to the basic techniques used to produce liposomes, various ancillary techniques have been developed for post-preparation treatment of liposomes to improve their properties. In particular, as discussed more fully below, many of the LUV techniques described above have required sizing of the finished liposomes by filtration using, for example, a series of polycarbonate filters. See Liposomes, supra, Ch. 1, pages 37-39, 45; and Szoka, et al., Biochimica et Biophysica Acta, 601:559 (1980). Series of polycarbonate filters have also been used to size MLVs. See F. Olson, et al., Biochimica et Biophysica Acta, 557:9 (1979), and Bosworth, et al., Journal of Pharmaceutical Sciences, 71:806 (1982).
- Although each of the foregoing techniques can be used to produce liposomes, none of these techniques are totally satisfactory. For example, each of the commonly used LUV techniques involves combining the components making up the liposome with a lipid solubilizing agent, i.e., either an organic solvent or a detergent. As is well known in the art, solvents and detergents can adversely effect many materials, such as enzymes, which one may want to encapsulate in liposomes, and thus these techniques cannot be used with these materials. Also, in applications such as the generation of drug carrier systems, the possible presence of these potentially toxic agents is undesirable.
- Moreover, these techniques often require lengthy dialysis procedures which can never completely remove the solvent or detergent employed. See, for example, Allen, et al., Biochimica et Biophysica Acta, 601:328 (1980). Further, a variety of protocols are required depending on the lipid species. For example, the limited solubility of certain lipids (e.g., cholesterol, phosphatidylethanolamine (PE), and phosphatidylserine (PS)) in ether or ethanol requires modification of techniques employing these solvents. Alternatively, detergent dialysis procedures employing non-ionic detergents such as octylglucoside are tedious to apply as they can involve several days of dialysis. Plainly, the need to separate lipid solubilizing agents from the finished liposomes materially decreases the usefulness of these methods.
- Along these same lines, the prior art LUV techniques have, in general, produced liposomes of various sizes, as well as aggregates of liposomes, thus requiring the additional step of sizing the finished liposomes with a series of filters. Again, this makes the overall process more time consuming and complicated.
- The fusion techniques include similar drawbacks. For example, the calcium ion/calcium chelating agent technique, like the solvent and detergent techniques, involves the use and subsequent removal of materials in addition to those actually making up the finished liposomes, in this case, the chelating agent and the added calcium ions. As with the solvents and detergents, these materials represent possible sources of contamination, limit the usefulness of the technique, and make the technique more complicated. Also, this technique requires that the composition of the liposomes include some phosphatidylserine.
- As to the freeze-thaw technique, this technique suffers from the drawback that the specific trapping capacity of the lipsomes produced by the technique drops off sharply at phospholipid concentrations above about 20 mg/ml.
- The SUV techniques have similar problems. For example, high energy sonication can cause oxidation and degradation of phospholipids and may damage solute molecules which one wants to capture in the interior space of the liposomes. Also, when performed using a sonication probe, high energy sonication can cause probe erosion, and if one with bath sonication in combination with radioactive materials, can produce a potentially hazardous aerosol. Low energy sonication is slow, can be destructive to phospholipid molecules, and cannot be used to prepare large quantities of liposomes. Further, the sonication approach results in low trapping efficiencies.
- The infusion type SUV procedures suffer the same problems as the LUV infusion procedures. The high pressure French press technique has its own set of problems, including difficulties in making the technique repeatable, the need for post-preparation filtration to remove those MLVs which have not been converted to SUVs, the need for expensive and cumbersome equipment capable of withstanding the high pressures used, and contamination of the product by disintegration of components of the apparatus which occurs during processing of the liposomes. See, for example, Bosworth, et al., Journal of Pharmaceutical Sciences, 71:806 (1982). Also, this technique can only produce small liposomes having a low trapping efficiency.
- Turning to the size distribution aspects of the invention, various procedures have been investigated in the past in an attempt to find a way to control both liposome size and distribution. Each of these procedures has fallen short of the mark in one way or another. For example, Ching-hsien Huang, in Biochemistry, 8:344 (1969), described a multi-step technique for producing a homogeneous population of small unilamellar liposomes (SUVs) which involved sonicating a lipid suspension in a buffer for 2) hours, centrifuging the resulting product at 105,OOOxg for 1 hour to remove undispersed lipid, filtering the supernatant through an extensively washed 0.1 pm Sartorius filter, subjecting the filtrate to molecular sieve chromatography on a Sepharose 4B column which had previously been saturated with the lipid suspension and washed and equilibrated with the buffer, and collecting the second fraction eluted from the column. Although this procedure did produce a population of liposomes having a defined size distribution, it was obviously complicated and time consuming to use, it only produced SUVs, and it ran the risk of chemically changing the liposomes or their contents during either the long term sonication or the exposure to the Sepharose@ 4B column.
- In an attempt to overcome some of the problems with the Huang technique, Barenholz, et al., Biochemistry, 16:2806 (1977), developed a technique in which high speed centrifugation was substituted for molecular-sieve chromatography. In accordance with this technique, a lipid dispersion in a buffer was sonicated for 30 minutes, centrifuged for 15-30 minutes at 100,000xg to remove large multilamellar liposomes and sonication probe particles, and the supernatant from the 100,000xg centrifugation was recentrifuged at 159,000xg for periods of time ranging from 1 to 4 hours depending on the lipids, buffer compositions and temperatures used. This latter centrifugation produced three zones, the top one of which contained the desired homogeneous population of liposomes and had to be carefully removed without picking up part of the adjacent second zone. Although this technique did eliminate the use of Sepharose 4B columns, it was still long and complicated, still only produced SUVs, and still had the problems arising, from sonication. Along these same lines, Watts, et al., Biochemistry, 17:1792 (1978), reported preparing a homogeneous population of SUVs from dimyristoylphosphatidylcholine (DMPC) by sonication followed by centrifugation at 105,000xg for 10 minutes at 4°C.
- In addition to the efforts directed at obtaining homogeneous populations of SUVs, numerous attempts have been made to obtain homogeneous populations of larger liposomes, i.e., liposomes having diameters larger than about 50 nm. The majority of these efforts have involved the use of a series of polycarbonate filters of decreasing pore size.
- For example, Olson, et al., in Biochimica et Biophysica Acta, 557:9 (1979), described the sequential extrusion of large multilamellar liposomes through polycarbonate filters having pore sizes of 1.0, 0.8, 0.6, 0.4 and 0.2 µm. See also Brendzel, et al., Biochimica etBiophysicaActa, 596:129 (1980). Olson's laboratory also reported the application of their technique to the sizing of large unilamellar liposomes prepared by reverse phase evaporation. See Biochimica et Biophysica Acta, 601:559 (1980). In this case, filters having pore sizes of 0.4, 0.2, 0.1, and 0.08 µm were used.
- Although the Olson work as reported in the literature would appear to produce unimodal populations of large liposomes (see, for example, Figures 1e and 3d in the 1979 article, and Figures 1D,2D, and 3D in the 1980 article), as described in detail in Example 10, infra, it has been surprisingly found that when quasi-elastic light scattering is used as the technique for determining size distributions, liposomes prepared by the Olson technique turn out not to have a unimodal distribution. In terms of large scale commercial production of liposomes, quasi-elastic light scattering is at present the only known real-time physical method for defining size distributions, so that in terms of commercial applications, the Olson procedure cannot be said to actually produce a population of liposomes which is unimodal.
- In addition to the Olson sequential polycarbonate filter approach, other techniques have been tried in the hope of obtaining a homogenous population of relatively large liposomes. For example, Schullery, et al., Chemistry and Physics of Lipids, 12:75 (1973), described the use of Millipore filters having pore sizes of 8.0, 1.2, 0.80, 0.65, and 0.45 pm to size large multilamellar phosphatidylcholine liposomes.
- Rhoden, et al., Biochemistry, 18:4173 (1979), reported the production of liposomes having diameters between 34 and 128 nm by solubilizing phosphatidylcholine and cholesterol in a sodium cholate solution and then removing that detergent by hollow fiber dialysis. The size of the liposomes was varied by adjusting the phospholipid/cholesterol ratios and the pH and ionic strength of the dialysate. It was observed that broader distributions were produced for larger liposomes.
- Boswoth, et al., Journal of Pharmaceutical Sciences, 71:806 (1982), combined the sequential polycarbonate filter sizing technique with dialysis across the same types of filters. Liposomes were prepared by mechanical agitation or by the French press techniuqe of Barenholz, et al., FEBS Lett., 99:210 (1979). Those produced by mechanical agitation were sized using filters having pore sizes of between 0.2 and 1.0 pm, with the liposomes being passed twice through the smallest filter and, in some cases, twice through each of the filters. For dialysis, filters having pore sizes between 0.05 and 3 µm were used.
- Enoch, et al., Proc. Natl. Acad. Sci. USA, 76:145 (1979), described the preparation of liposomes having diameters of 100 nm by detergent treatment of sonicated vesicles followed by gel filtration on Sepharose 4B. Hamilton, et al., Journal of Lipid Research, 21:981 (1980), described the preparation of liposome populations of various sizes using a French press in combination with ultracentrifugation and gel chromatography on columns of 2% or 4% agarose. Reeves, et al., J. Cell. Physiol., 73:49 (1969), reported the production of a population of giant liposomes (mode=1,200 nm) having a log-normal distribution, but vesicles smaller than 1000 nm were measured with difficulty, and those smaller than 500 nm were not measured at all.
- A review of some of the foregoing procedures can be found in Szoka, et al., Ann. Rev. Biophys. Bioengr., 9:467, 493-494 (1980). See also Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983,
Chapter 1. - In view of the foregoing state of the art, it is evident that there is a substantial and continuing need for an improved method for preparing unilamellar liposomes of both the SUV and LUV types.
- Moreover, it is also evident that since at least as early as 1969, there has been a continuing effort to produce populations of liposomes having defined size distributions. Much of that effort has been directed towards obtaining populations having mean diameters greater than about 50 nm. Along with the desire for the populations per se, there has been a parallel desire for a generally applicable and simple to use technique which will reproducibly generate populations of the type desired for a wide variety of processing conditions.
- Accordingly, it is one of the objects of the present invention to provide an improved technique for producing unilamellar liposomes. More particularly, it is an object of this invention to provide a simple, reproducible technique for producing unilamellar liposomes which can be performed with readily available and relatively inexpensive equipment, which has a minimum number of steps, which has a high output of liposomes per unit time, and which does not require that the components making up the liposomes be sonicated or combined with solvents, detergents or other extraneous materials.
- It is another object of the invention to provide a technique for producing liposomes of both the unilamellar and multilamellar types which does not require the use of solvents, detergents or other extraneous materials.
- It is a further object of the invention to provide populations of liposomes having defined size distributions. It is an additional object of the invention to provide a straightforward method for obtaining such populations.
- To achieve the foregoing and other objects, the invention, in accordance with certain of its aspects, provides a method for producing a population of substantially unilamellar liposomes which involves repeated extrusions at moderate pressures of previously formed liposomes through a filter having a pore size below a critical upper limit, specifically, below approximately 100 nm.
- In this manner, the invention provides a variety of advantages over previously known systems for producing unilamellar liposomes, including the following: 1) the ability to form unilamellar vesicles from a wide range of lipids; 2) the ability to use high lipid concentrations (e.g., on the order of 300 umol/ml) so as to easily achieve high trapping efficiencies; 3) the ability to provide reproducible and very rapid production of unilamellar vesicles, and, in particular, large unilamellar vesicles, through the use of high extrusion flow rates and automatic or semi-automatic recycling of the liposomes through the filter; 4) the ability to produce liposomes of a desired size by using a single pore size filter with minimum filter clogging problems; 5) the ability to avoid the use of organic solvents and detergents; and 6) the ability to provide an overall relatively gentle process.
- In some cases, rather than completely transforming a population of multilamellar liposomes into a population of substantially unilamellar liposomes, it is desirable to only partially decrease the lamellarity of the population without reaching the fully unilamellar stage. Filters having a pore size of about 100 nm are still used in accordance with this aspect of the invention, but with a reduced number of passes through the filter.
- In accordance with some of its other aspects, the invention provides a method for producing liposomes directly from a lipid powder or pellet by simply combining the powder or pellet with an aqueous buffer and then applying sufficient pressure to the lipid/buffer mixture to repeatedly pass it through a filter. If the filter has a pore size less than about 100 nm, substantially unilamellar liposomes are produced. If the filter has a pore size significantly above 100 nm, e.g., on the order of 200 nm, multilamellar lipsomes are produced. Significantly, in either case, the liposomes are completely solvent free, in that, not even chloroform, as has been used in the past to produce MLVs, is required for liposome production in accordance with these aspects of the present invention.
- In accordance with further of its aspects, the invention provides populations of liposomes having essentially unimodal distributions about mean diameters which are greater than 50 nm.
- In accordance with still further of its aspects, the invention provides a method for increasing the homogeneity of a population of liposomes by repeatedly passing the liposomes through one or more filters of a constant pore size until the size distribution of the population becomes essentially unimodal.
- The attainment of the foregoing and other objects and advantages of the present invention is described fully below in connection with the description of the preferred embodiments of the invention.
- Figures 1A and 1 B are schematic diagrams of apparatus suitable for practicing the present invention. In Figure 1A, the liposome suspension is recycled through the filter by hand, while in Figure 1 B, the recycling has been partially automated.
- Figure 2 shows the 31p NMR signal intensity arising from egg phosphatidylcholine (EPC) multilamellar vesicles (in the presence of 5 mM MnCl2) as a function of the number of extrusions through polycarbonate filters with 100 nm (circles) and 200 nm (squares) pore sizes. The error bars represent standard deviations (n=6 for the point at 10 extrusions through the 100 nm filter; n=3 for the point at 30 extrusions). All other experimental points represent the average obtained from two separate experiments. The lipid concentration in all cases was between 30 and 60 umol/ml.
- Figure 3 shows four freeze-fracture micrographs of vesicles prepared by repeated extrusion of multilamellar vesicles of varying lipid composition through polycarbonate filters: (a) soya phosphatidylcholine (PC) MLVs extruded through a 100 nm filter; (b) soya PC-soya PS (1:1) MLVs extruded through a 100 nm filter; (c) soya PE-soya PS (1:1) MLVs extruded through a 100 nm filter; (d) soya PC MLVs extruded through a filter with a 200 nm pore size. The arrow in part (d) indicates a cross fracture revealing inner lamellae. All micrographs have the same magnification and the direction of shadowing is indicated by the arrowhead in the bottom right corner of each section. In each case, the extrusion procedure was repeated 10 times on lipid systems containing 40-70 umol/ml phospholipid.
- Figure 4 shows the size distribution of soya PC vesicles obtained after 10 extrusions through a polycarbonate filter with a 100 nm pore size. The vesicle diameters were measured from freeze fracture micrographs employing the technique of van Venetie et al., (1980) J. Micros., 118:401-408. The black and half-tone columns represent vesicles that did and did not undergo freeze-thaw cycling, respectively.
- Figure 5 shows the calorimetric behaviour of hydrated dipalmitoylphosphatidylcholine (DPPC) in large multilamellar vesicles (MLVs) and in large unilamellar vesicles prepared by the extrusion technique of the present invention (LUVETs). The MLVs were formed by vortexing a dry lipid film in the bottom of a test tube in the presence of a NaCl buffer at 50°C, whereas the LUVETs were formed by repetitive extrusion (10 times) of the MLVs (50 mg lipid/ml) through 100 nm pore size polycarbonate filters at 50°C. Scan rates of 2.0°K/min were employed.
- Figure 6 shows the trapping efficiency as a function of lipid concentration for liposomes prepared in accordance with the present invention both with (open circles) and without (closed circles) freeze-thawing. 14C-inulin was used as a trap marker.
- Figure 7 shows the 31p NMR signal intensity arising from egg phosphatidylcholine (EPC) multilamellar vesicles (in the presence of 5 mM MnC12) as a function of the number of extrusions through polycarbonate filters with 50 nm (open circles) and 30 nm (solid circles) pore sizes. The lipid concentration in all cases was 100 mg/ml.
- Figures 8 and 9 show freeze-fracture micrographs of the vesicles of Figure 7 processed through 50 nm and 30 nm filters, respectively. In each case, the upper portion of the figure (Figures 8A and 9A) was prepared after one extrusion (x1) through two stacked polycarbonate filters, and the lower portion (Figures 8B and 9B) after ten extrusions (x 10).
- Figure 10 shows the clearance of 125|-tyraminyl-inulin (125|T|) entrapped in egg phosphatidylchloline (PC)-cholesterol (1:1) LUVETs from the rat circulation (circles) and subsequent excretion in the urine (squares). The LUVETs were prepared in accordance with the present invention and were injected into the tail vein of 150-175 g female Wistar rats at a dose level of 0.5 umol phospholipid in 100 µl HBS. Urine was collected in metabolic cages. Blood was withdrawn and the animals sacrificed at the indicated times and the total amount of 125 ITI in the blood calculated assuming 4.9 ml blood per 100 g rat. Results are expressed as percentages of the total 125|T| injected ±s.e. (n=4).
- Figure 11 shows the long term tissue distribution of the LUVETs of Figure 10. The symbols correspond to liver (circles); carcass (triangles) and spleen (squares). Results are expressed as percentages of total 125 ITI in vivo (total 125|T| injected minus amount excreted) ±s.e. (n=4).
- As described above, the present invention provides extrusion techniques for producing: 1) populations of substantially unilamellar liposomes; and 2) populations of liposomes having substantially unimodal distributions (hereinafter referred to as the "unilamellar" and "unimodal" aspects of the invention, respectively). In addition, the invention allows liposomes to be produced without the use of any solvents, detergents or other extraneous materials (hereinafter referred to as the "solvent free" aspects of the invention).
- The populations of substantially unilamellar liposomes are produced by subjecting previously formed liposomes to multiple extrusions at moderate pressures through a filter having a pore size of less than or equal to about 100 nm.
- The previously formed liposomes can have a variety of compositions and can be prepared by any of the techniques now known or subsequently developed for preparing liposomes.
- For example, the previously formed liposomes can be formed by the conventional technique for preparing MLVs, that is, by depositing one or more selected lipids on the inside walls of a suitable vessel by dissolving the lipids in chloroform and then evaporating the chloroform, adding the aqueous solution to be encapsulated to the vessel, allowing the aqueous solution to hydrate the lipid, and swirling or vortexing the resulting lipid suspension to produce the desired liposomes. This technique employs the most gentle conditions and the simplest equipment and procedures known in the art for producing liposomes. Also, this technique specifically avoids the problems with sonication or the use of detergents, solvents (other than chloroform) or other extraneous materials, discussed above.
- Alternatively, in accordance with the solvent free aspects of the present invention, the liposomes which are to be repeatedly extruded through the filter can be prepared by simply mixing a lipid powder or pellet and buffer together and then directly extruding that mixture through the filter. If the filter has a pore size of less than about 100 nm, this procedure produces unilamellar liposomes, while if the pore size is substantially greater than 100 nm, multilamellar liposomes are produced. In either case, the procedure eliminates the use of all solvents, including chloroform.
- With regard to the production of populations of liposomes having substantially unimodal distributions, the liposomes making up the population can have a variety of compositions and can be in the form of multilamellar, unilamellar, or other types of liposomes or, more generally, lipid-containing particles, now known or later developed. For example, the lipid-containing particles can be in the form of steroidal liposomes, stable plurilamellar liposomes (SPLVs), monophasic vesicles (MPVs), or lipid matrix carriers (LMCs) of the types disclosed in copending and commonly assigned U.S. Patent No. 4,522,803; No. 4,588,578; No. 4,610,868 respectively.
- The mean diameter of the population will depend on the manner in which the liposomes are to be used. For example, as recognized by persons skilled in the art, for diagnostic applications, mean diameters in the range of 100 nm to 500 nm are generally preferred, while for depoting of drugs, larger diameters, e.g., on the order of 500 nm to 1000 nm, are preferred, and for applications where endocytosis is desirable, smaller diameters, e.g., on the order of 50 nm to 100 nm, are preferred. Similar ranges have been recognized in the art for other applications. See, for example, Liposomes, supra.
- Mean diameters of populations of liposomes can be measured by various techniques known in the art, including freeze-fracture and quasi-elastic light scattering. As discussed above and in more detail below, quasi-elastic light scattering is preferred in the context of the unimodal aspects of the present invention, and the values for liposome diameters reported herein in connection with those aspects were measured using this technique.
- The substantially unimodal population of liposomes is prepared by repeatedly passing previously formed liposomes through filters of a constant pore size until the size distribution of the population in fact becomes unimodal. It has been surprisingly found that repeated passages through filters of a constant pore size causes bimodal aspects of the original size distribution of the liposomes, as well as any higher modal aspects, to eventually disappear. For the method to work, however, it is necessary to use one pore size, and use it repeatedly.
- The previously formed liposomes can be prepared by any of the techniques now known or subsequently developed for preparing liposomes. For example, the previously formed liposomes can be formed by the conventional technique, discussed above, for preparing multilamellar liposomes (MLVs).
- Alternatively, techniques used for producing large unilamellar liposomes (LUVs), such as, reverse-phase evaporation, infusion procedures, and detergent dilution, can be used to produce the previously formed liposomes. A review of these and other methods for producing liposomes can be found in the text Liposomes, supra, the pertinent portions of which are incorporated herein by reference. As other alternatives, the previously formed liposomes can be produced in accordance with the procedures described in U.S. Patent No. 4,522,803; No. 4,588,578; No. 4,721,612. Also, rather than using liposomes per se, other lipid-containing particles, such as those described in U.S. Patent No. 4,610,868 referred to above, can be used in the practice of the present invention. In such cases, the resulting unimodal population will, in general, not be a population of liposomes, but rather a population having similar characteristics to those of the original lipid-containing particles used.
- In choosing a technique for producing the previously formed liposomes, it is important to select one that will not produce a substantial number of liposomes having a diameter significantly smaller than the pore size selected for generating the unimodal population. Otherwise, it may take an extremely high number of passes through the filter to incorporate the small liposomes into the unimodal population. Since size distributions for populations of liposomes are relatively easily determined, selecting a technique which satisfies this requirement is well within the skill of persons skilled in the art.
- Rather than using previously formed liposomes as the starting material, if desired, the substantially unimodal population of liposomes can be prepared using the solvent free approach, discussed above. That is, the population of liposomes can be prepared directly from a lipid powder or pellet and buffer by simply mixing these ingredients together and then directly passing that mixture through filters of a selected pore size a sufficient number of times to achieve the desired unimodality.
- The filter used to generate the unilamellar liposomes or the unimodal distribution of liposomes is preferably of the type which has straight through channels. Polycarbonate filters of this type produced by Nuclepore, Inc., Pleasanton, CA, have been found to work successfully in the practice of the present invention. In a typical procedure, the filter may have to be changed after the first two or three passes of the liposome suspension due to pore clogging. Clogging in general depends on such variables as lipid composition, purity and concentration, as well as on the pressure and flow rates used.
- The most critical parameter in preparing unilamellar liposomes in accordance with the present invention is the size of the filter's channels. It has been found that unilamellar liposomes cannot be produced from multilamellar liposomes, no matter how many times the MLVs are passed through the filter, if the filter's pore size is significantly above about 100 nm, e.g., if the pore size is about 200 nm (see Example 2, infra). Accordingly, only filters having a pore size equal to or below about 100 nm can be used in connection with this aspect of the invention.
- As illustrated in Example 6 below, the size of the unilamellar liposomes produced depends on the pore size of the filter used, the mean diameter being, in general, somewhat smaller than the pore size. If desired, the liposome's mean diameter, as well as their trapped volume (ul per umol phospholipid), can be easily increased using the freeze-thaw procedure discussed above (see also Example 4, infra). Importantly, since this procedure does not involve the use of solvents, detergents or other extraneous materials, the increase in liposome size is not at the expense of introducing contamination and degradation problems. Vesicle size and trapped volumes can also be manipulated by varying other parameters of the system, such as, lipid composition.
- The number of passes through the filter needed to produce the desired unilamellar liposomes depends on the filter characteristics (pore size, composition and geometry) and the materials from which the liposomes are to be made. As illustrated by Example 2, infra, five or more passes through a double stacked polycarbonate filter having a pore size of 100 nm are typically required to obtain unilamellar liposomes. Less passes may be needed for smaller pore sizes. For example, with 30 nm and 50 nm filters, two to four passes are in general sufficient to produce a substantially unilamellar population of liposomes (see Example 6, infra). Also, if the goal is only to reduce the lamellarity of the population, rather than to achieve substantial unilamellarity, less passes are needed. The appropriate number of passes for any particular system can easily be determined by persons of ordinary skill in the art by simply sampling the finished liposomes to determine when the desired degree of lamellarity has been achieved.
- With regard to the unimodal aspects of the invention, the pore size of the filter is the primary determinant of the mean diameter of the final population. In general, within approximately 15 to 50 percent, the mean diameter is approximately equal to the pore size. However, for pore sizes below about 100 nm, the mean diameter of the population tends to level off at about 75 nm as measured by quasi-elastic light scattering, irrespective of the specific pore size used. As discussed above, for pore sizes below about 100 nm, the finished liposomes are found to be substantially unilamellar, irrespective of the lamellarity of the original population. For pore sizes above 100 nm, multilamellar liposomes remain multilamellar and unilamellar liposomes remain unilamellar.
- The number of passes through the filter needed to produce a substantially unimodal population depends on the filter characteristics (pore size, composition and geometry) and the materials from which the liposomes are to be made. In some cases three to five passes through double stacked polycarbonate filters are sufficient to produce a unimodal population. As a general proposition, 25 passes through double stacked polycarbonate filters will produce the desired unimodal distribution for most liposome preparations. The appropriate number of passes for any particular system can easily be determined by persons of ordinary skill in the art by simply sampling the finished liposomes to determine when substantial unimodality has been achieved.
- Passage of the liposomes through the filter to produce the unimodal, unilamellar, or both unimodal and unilamellar population of liposomes is accomplished under pressure. Pressures of various magnitudes can be used depending upon the type and composition of the liposomes to be produced, the specific characteristics of the equipment employed, and the rate at which liposomes are to be produced.
- Maximum pressures generally are limited by the pore size of the support used to hold the filter. For a filter support having a pore size of about 30 pm, pressures between about 100 and 700 psi (689.47 and 4,826.29 kPa have been found to work successfully. These pressures produce intact liposomes, give high flow rates (on the order of 20-60 ml/min for a double stacked polycarbonate filter having a size of 100 nm), and produce homogeneous size distributions, e.g., 60-100 nm diameter liposomes for a 100 nm filter. With a filter support having a pore size smaller than 30 pm, higher pressures can be used.
- As with the number of passages through the filter, the appropriate pressure for a particular system can be readily determined by persons skilled in the art by examining the finished liposomes to determine if they are substantially intact and have the desired unimodal distribution and/or unilamellarity.
- Pressures in the 100-700 psi (689.47-4,826.29 kPa) range are also preferred because they allow for the extrusion of solutions having lipid concentrations on the order of about 300 umols phospholipid per ml without significant filter clogging. Prior art liposome sizing techniques employing polycarbonate filters used pressures less than 100 psi (689.47 kPa), and thus were limited to lipid concentrations of 60 umol/ml. The use of high lipid concentrations has resulted in trapping efficiencies on the order of 30% for the present invention. Rapid extrusion rates on the order of 20 ml/min and above are still achieved for such high lipid concentrations when pressures in the range of 300-500 psi (2,068.41-3,447.35 kPa) are used.
- Flow rates on the order of 20-60 ml/min do not represent the maximum flow rates that are achievable with the present invention, but merely represent rates consistent with convenient collection of the extruded material. Maximum flow rates are sensitive to the concentration of lipid, the history of the sample (i.e., whether it has been extruded one or more times), the pressure employed, and the nature of the lipids themselves. For example, "gel state" lipids cannot be extruded. Such lipids (e.g., dipalmitoyl phosphatidylcholine (DPPPC)) must be heated above their gel to liquid crystalline transition temperature (41°C for DPPC) before the extrusion process can proceed.
- At increased pressure, the extrusion can be exceedingly rapid. For example, a 5 ml dispersion of 50 mg/ml egg phosphatidylcholine (EPC) has been extruded through a 100 nm pore size filter in less than 2 seconds at 500 psi (3,447.35 kPa), corresponding to a flow rate of at least 150 ml/min. This rate would be increased further by higher pressure or temperature.
- Suitable apparatus for practicing the present invention is shown in Figures 1A and 1B. As shown in Figure 1A,
liposome suspension 4 which is to be extruded throughfilter 6 is introduced intopressure chamber 2 by means ofinjection port 8. The injection port also serves as a release valve.Pressure chamber 2 is formed fromupper portion 10 andlower portion 12 which are connected together by, for example,bolts 14. A seal between these portions andfilter 6 is provided by O-ring 16. Preferably, the chamber is made of clear plastic so that the extrusion of the suspension from the chamber can be visually observed. The filter is supported withinchamber 2 byfilter support 18. In practice, it has been found convenient to use two stacked polycarbonate filters to formfilter 6. - Pressure is supplied to
chamber 2 by means ofconduit 20 which is connected to a source of pressure, e.g., a high pressure nitrogen tank (not shown).Conduit 20 includes valve andregulator 22 for adjusting the pressure withinchamber 2. The material extruded fromfilter 6 is removed fromchamber 2 by means ofconduit 24 and collected in receivingvessel 26. In practice,valve 22 is closed prior to the time all of the suspension has been extruded fromchamber 2 so as to prevent high pressure gas from flowing through the system and blowing the suspension out of receivingvessel 26. After having been collected invessel 26, the extruded material is repeatedly returned tochamber 2 by means ofinjection port 8, until the original population of liposomes has passed through filter 6 a sufficient number of times so as to substantially increase its unimodality and/or unilamellarity. - Figure 1B shows a modification of the apparatus of Figure 1A wherein the recycling of the extrudate is partially automated. The apparatus shown in this figure is used as follows.
- First, a
filter 6 is installed in the apparatus by removing threadedretainer plug 5, filtersupport housing 9, and 0-ring 16 fromaluminum housing 7. A filter is then placed on the filter support and the components reassembled withplug 5 being tightened until 0-ring 16 is compressed againstinner plexiglas housing 11 contained withinouter aluminum housing 13. If desired, a porous drain disc (not shown) can be placed under the filter. - A sample is then loaded into receiving
chamber 3 by rotating load/recycle/discharge valve 15 until load/discharge tube 17 is aligned withinlet port 19, and by rotating pressure/vent valve 23 untilvent port 25 is aligned withexhaust port 21. A sample can then be introduced into the receiving chamber through load/discharge tube 17. Most conveniently, this is done by attaching a short length of flexible tubing and a hypodermic syringe to load/discharge tube 17. - Once the sample has been completely loaded into receiving
chamber 3, it is transferred to pressurechamber 2 by depressingtransfer valve 27. The sample is now ready for extrusion throughfilter 6. To perform the extrusion, pressure/vent valve 23 is rotated untilgas inlet port 31 is aligned withpressure port 33, and load/recycle/discharge valve 15 is rotated to a position where recycleport 37, formed invalve 15, is aligned at one end withinlet port 19 and at the other end withbypass port 29.Gas inlet port 31 is connected to threadedaperture 35 which serves to connect the apparatus to a valved and regulated external source of high pressure gas, e.g., a valved and regulated high pressure nitrogen tank (not shown). - Pressure is then applied to
pressure chamber 2 causing the sample to pass from that chamber to receivingchamber 3 by way offilter 6, bypassport 29, recycleport 37, andinlet port 19. This accomplishes one extrusion of the sample through the filter. Flow frompressure chamber 2 to receivingchamber 3 can be visually observed throughplexiglas housing 11, and the amount of pressure applied can be adjusted to achieve a gentle flow. - Once all of the sample has been transferred to receiving
chamber 3, the valve on the external source of pressure is closed, and pressure/vent valve 23 is rotated to first bringvent port 25 into alignment withexhaust port 21 and then into alignment withpressure port 33, thus venting both receivingchamber 3 andpressure chamber 2. The sample can now be reintroduced to pressurechamber 2 by simplydepressing transfer valve 27. With the sample inpressure chamber 2, the extrusion process is repeated following the procedures described above for the first extrusion. Note that with the sample in the receiving chamber and with both chambers having been vented, a new filter can be installed, if desired, following the procedures described above. - Once the receiving chamber-pressure chamber-receiving chamber cycle has been repeated the desired number of times, the sample is discharged from the apparatus through load/
discharge tube 17 by first aligninggas inlet port 31 withexhaust port 21 and load/discharge tube 17 withinlet port 19, and then applying pressure to the system from the external pressure source. In practice, the external pressure is shut off before all of the sample has leftchamber 3 to avoid high gas flows at the end of the evacuation. Rather than using load/discharge tube 17, the sample can also be removed by disassembling threadedretainer plug 5, filtersupport housing 9, and 0-ring 16 fromaluminum housing 7, and then depressingtransfer valve 27 to cause the sample to flow into the pressure chamber and out the bottom of the apparatus. - If desired, the apparatus shown in Figure 1B, or equivalent apparatus, can be equipped with conventional automatic fluid handling equipment and controls to achieve a completely automated process. Also, so as to be able to maintain the temperature of the sample above the gel to liquid crystalline transition temperature of the lipids used, the pressure chambers of whatever equipment is employed can be heated using a water jacket or similar device.
- Without intending to limit it in any manner, the present invention will be more fully described by the following examples. The materials and methods which are common to the various examples presented below are as follows.
- Egg phosphatidylcholine (EPC) and soya phosphatidylcholine (SPC) were isolated employing standard procedures (Singleton, et al., Journal of the American Oil Chemical Society, 42:53 (1965)). Corresponding varieties of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were prepared from EPC and SPC to produce EPE, SPE, EPS and SPS (see Comfurius, P. and Zwaal, R. F. A. (1977) Biochim. Biophys. Acta, 488:36-42). The lipids from the soya source are considerably more unsaturated than those derived from EPC, due to the high content of linoleic acid in SPC (see Tilcock, C. P. S. and Cullis, P. R. (1981) Biochim. Biophys. Acta, 641:189-201). All lipids were more than 95% pure as determined by TLC. Acidic phospholipids (PS) were converted to the sodium salt form as described in Hope, M. J. and Cullis, P. R. (1980) Biochim. Biophs. Res. Commun., 92:846-852. Cholesterol (Sigma, St. Louis) was used without further purification.
- To determine trapped volumes, multilamellar vesicles were prepared in accordance with the procedures described in Example 1, infra, but in the presence of 1 uCi of 22Na or 14C-inulin (NEN, Canada). Unilamellar liposomes were then prepared from the multilamellar liposomes, again following the procedures of Example 1.
- An aliquot (100 ul) was then loaded onto a SephadexO G-50 column packed in a 1 ml disposable syringe, and vesicles eluted by centrifugation of the column at 500 g for 3 min. See Pick, U. (1981) Arch. Biochim. Biophys., 212:186-194. In the case of 22Na this was sufficient to remove all the untrapped material. However, to remove all the untrapped inulin this procedure was either repeated once more or a single pass through an Ultragel column (LKB-ACA34) was employed. Aliquots of the eluted material were assayed for lipid phosphorus according to the method of Bottcher, C. J. F., Van Gent, C. M. and Pries, C. (1961) AnaL Chim. Acta, 24:203-204; trapped 22Na was determined employing a Beckman 8000 gamma counter and trapped 14C inulin was estimated using a Phillips PW-4700 liquid scintillation counter. Trapped volumes were then calculated as ul of trapped volume per umol of phospholipid.
- 31P NMR was employed to provide an indication of the extent to which the vesicle preparations were unilamellar. Specifically, Mn2+ was added to the vesicle dispersion (3 ml, 30-60 umol phospholipid per ml in a 15 mm diameter NMR tube) at levels (5 mM) sufficient to broaden beyond detection the 31P NMR signal from those phospholipids facing the external medium. If the vesicles are large and unilamellar, then approximately 50% of the signal should remain following the addition of Mn2+. The impermeability of the vesicles to Mn2+ was demonstrated by following the timecourse of the signal intensity, which for the PC systems investigated was found to be stable over a period of days. Spectra were obtained employing a
Bruker WP 200 NMR spectrometer operating at 81 MHz. Accumulated free induction decays corresponding to 1000 transients were collected using a 15 usec 90° radiofrequency pulse, gated proton decoupling and a 20 KHz sweep width. An exponential multiplication corresponding to a 50 Hz linebroadening was applied prior to Fourier transformation. Signal intensities were measured by cutting out and weighing spectra with triphenylphosphite (in a small central capillary in the NMR tube) as a reference. - Vesicle size distributions for Examples 1-7 and part of Example 8 were determined by freeze-fracture. Vesicle preparations were mixed with glycerol (25% by volume) and frozen in a freon slush. Samples were fractured and replicated employing a Balzers BAF 400D apparatus, and micrographs of replicas were obtained using a Phillips 400 electron microscope. Vesicle size distributions were determined by measuring the diameter of fractured vesicles that were 50% shadowed according to the procedure of van Venetie et a/., (1980) J. Micros., 118:401-408.
- Vesicle size distributions for part of Example 8 and Examples 9-10 were determined using quasi-elastic light scattering, also known as dynamic light scattering or photon-correlation spectroscopy.
- As known in the art, this technique involves passing coherent light, e.g., light produced by a helium- neon laser, through a sample of the suspended vesicles and measuring the time dependent fluctuations in the intensity of the light scattered by the vesicles. An autocorrelation function is then calculated from this data. As can be shown theoretically, this autocorrelation function is directly related to the diffusion coefficients of the vesicles in the sample, which in turn are a function of the hydrodynamic radii of the vesicles. Accordingly, different vesicle size distributions will produce different autocorrelation functions.
- In practice, unique particle size distributions are not obtained directly from autocorrelation functions. Rather, a distribution is assumed for the vesicles, and a determination is then made as to how well the autocorrelation function calculated from the measured data fits the autocorrelation function that would be produced if the vesicles in the sample actually had the assumed distribution.
- Specifically, if it is assumed that the intensity-weighted distribution of the diffusion coefficients of the vesicles is a unimodal Gaussian distribution, then it can be shown theoretically that a second order polynomial, i.e., a polynomial in powers of t up to t2, will exactly fit the natural logarithm of the autocorrelation function. See D. E. Koppel, Journal of Chemical Physics, 57:4814 (1972). Accordingly, the extent to which a second order polynomial, i.e., a quadratic, in fact does fit the natural logarithm of the autocorrelation function for a particular sample is an accurate measure of the extent to which the diffusion coefficients of the vesicles in the sample have a unimodal Gaussian distribution. As used in the art, this procedure is often referred to as a cumulants analysis of the autocorrelation function.
- As known in the art, it is a straightforward matter to 1) determine the natural logarithm of an autocorrelation function, 2) to fit a second order polynomal to the natural logarith, and 3) to determine the goodness of fit of that polynomial to that logarithm. Accordingly, the Gaussian distribution approach is at present the most practical way to characterize and compare populations of vesicles, and thus it is the approach used herein in connection with the unimodal aspects of the present invention.
- Specifically, in accordance with those aspects, a population of liposomes is considered to be substantially "unimodal" when the logarithm of its autocorrelation function fits closely to a second order polynomial. In the examples presented below, autocorrelation functions were obtained using a
Nicomp Model 200 Laser Particle Sizer (Nicomp Instruments, Inc., Santa Barbara, California). This equipment uses the standard method of least-squares for curve-fitting and reports goodness of fit as a chi2 value derived from the deviations between the logarithm of the autocorrelation function and the values predicted by the second order polynomial. Values of chi2 in the range of 0-2 indicate a good fit of the data by the assumed unimodal Gaussian distribution, while high values of chi2 indicate a poor fit. - For a good fit, an estimate of the standard deviation of the distribution can be derived from the square root of the coefficient of the second order term of the polynomial. For distributions having a good fit, this estimate is reported in the examples, while for poor fits, the estimate is not reported, since, although a value can be calculated, such a value does not in fact serve as an estimate of the standard deviation.
- Inulin, periodic acid, sodium-m-arsenite, tyramine, G-25 Sephadex, sodium cyanoborohydride, sodium borohydride, and cholesterol were obtained from Sigma. Ultrogel® Ac34 was obtained from Pharmacia, carrier free Na125|3.7x 109 Bq/ml (100 mCi/mi) was supplied by Amersham and iodogen was obtained from Pierce. All other chemical were of analytical grade.
- This example illustrates the preparation of large unilamellar liposomes using the extrusion method of the present invention. For ease of reference, liposomes prepared in accordance with this technique are referred to herein by the acryonym "LUVETs", i.e., Large U nilamellar Vesicles by Extrusion Techniques.
- Large multilamellar vesicles (MLVs) were prepared by the conventional process as follows. First, lipid dissolved in chloroform was dried down and deposited as a film on the inside of a test tube. The MLVs were then formed by simply adding an aqueous buffer of 150 mM NaCl, 20 mM HEPES, pH 7.5, to the test tube and hydrating the lipid by vortex mixing.
- The resulting MLV dispersion (2-10 ml) was then transferred into the pressure chamber of the apparatus shown in Figure 1A, equipped with two stacked standard 25 mm polycarbonate filters having a 100 nm pore size (Nuclepore, Inc., Pleasanton, California, Catalog # 110605). Nitrogen pressure was applied to the chamber via a standard gas cylinder fitted with a high pressure (0-4000 psi (2,757.88 kPa)) regulator. The vesicles were extruded through the filter employing pressures of 100-700 psi (689.47-4,826.29 kPa) resulting in flow rates of 20-60 ml/min, and were collected and re-injected. Repetition of the extrusion procedure five or more times resulted in the production of large unilamellar liposomes having diameters of approximately 70 nm as measured by freeze fracture. The overall extrusion process including recycling generally took fifteen minutes or less.
- The following examples describe in detail the size, unilamellarity, trapped volume, trapping efficiency and influence of various lipid compositions on liposomes produced by the foregoing procedure. Also, the effects of a freeze-thaw procedure on trapped volume and the criticality of filter pore size are illustrated.
- This example demonstrates the criticality of filter pore size in producing unilamellar liposomes, and, in particular, the criticality of using a filter having a pore size of less than or equal to about 100 nm.
- EPC MLVs were prepared in accordance with the procedures described in Example 1 and then repeatedly passed through polycarbonate filters having pore sizes of 100 and 200 nm. The unilamellarity of the resulting liposomes was determined using the 31p NMR technique described above. The results are shown in Figure 2.
- As shown in that figure, for vesicles passed through the 200 nm filter, the signal intensity drops to approximately 65% after five passes through the filter and then remains relatively constant. For the 100 nm filter, on the other hand, the signal drops to approximately 50% after five or more passes.
- Since a drop in signal intensity to about 50% indicates that the liposomes are substantially unilamellar, while a drop to only 65% indicates substantial multilamellarity, these results show that the 100 nm filter succeeds in producing unilamellar liposomes, as desired, while the 200 nm filter continues to produce significant amounts of multilamellar liposomes irrespective of the number of passes through the filter.
- This conclusion is confirmed by the freeze-fracture micrographs shown in Figure 3. As shown in that figure, vesicles formed from SPC, SPC-SPS (1: 1) and SPE-SPS (1: 1) (Figures 3(a), (b) and (c), respectively) using a 100 nm filter do not exhibit a significant number of cross-fractures (less than 0.1 %) indicating the absence of inner lamellae. In contrast, cross-fractures are readily observable for SPC vesicles processed through a 200 nm filter (Figure 3(d)).
- These results clearly establish that in accordance with the present invention, unilamellarity depends upon the use of a filter having a pore size on the order of 100 nm or below.
- This example demonstrates the procedures of the present invention when used with 100 nm filters reproducibly result in the production of a relatively homogeneous population of LUVs for a variety of lipid constituents. Vesicle diameters and trapped volumes were determined by the methods described above. The results are shown in Figure 4 and Table I, infra.
- The half-tone columns in Figure 4 show the vesicle diameters measured for SPC LUVETs which were prepared by passing MLVs prepared in accordance with Example 1 through two (stacked) 100 nm pore size filters ten times. Table I shows in summary form the measured mean diameters and mean trapped volumes for this and other lipid compositions. As a control, EPC LUVs were prepared by two procedures (octylglucoside detergent dialysis and reverse phase evaporation) which are generally accepted to produce unilamellar vesicles, and the LUVs so produced were then extruded ten times through two (stacked) 100 nm pore size filters. See Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C. and Reynolds, J. A. (1981) Biochemistry, 20:833-840 and Szoka, F. and Papahadjopoulos, D. (1980) Ann. Rev. Bioeng., 9:467-508. The results for these controls are shown in Table I. (It is of interest to note with regard to the generality of the present invention that when the octylglucoside procedure was employed to make vesicles consisting of EPC/cholesterol (1:0.25), multilamellar vesicles were formed, whereas with the procedure of the present invention and the same lipid constituents, substantially unilamellar vesicles were formed).
- The vesicle diameter distribution shown in Figure 4 can be used to determine calculated values for trapped volumes and amount of inner monolayer phospholipid by assuming 1) an area per phospholipid molecule, e.g., 0.6 nm2 (see Schieren, H., Rudolph, S., Finkelstein, M., Coleman, P. and Weissman, G. (1978) Biochim. Biophys. Acta., 542:137-153); 2) a bilayer thickness, e.g., 4 nm (see Blaurock, A. E. (1982) Biochim. Biophys. Acta., 650:167-207); and 3) that the vesicles are unilamellar. These calculated values can then be compared with the experimentally observed trapped volumes and amounts of inner monolayer phospholipid to determine the proportion of unilamellar vesicles present.
- Following this approach for the vesicle size distribution shown in Figure 4 (half tone), it was determined that such a vesicle population (if unilamellar) would have an "inner monolayer" signal intensity (after the addition of Mn2+) of approximately 43% of the original intensity and that the trapped volume would be approximately 1.6 ul/umole. This is in reasonable agreement with the measured values of sequestered phospholipid (48%) and trapped volume (1.2±0.2 ul/umol) in view of the number of assumptions made, and, in particular, in view of the difficulty in estimating the area per phospholipid molecule which can greatly affect the trapped volume expressed as ul trapped per umole of phospholipid.
- Comparing the calculated trapped volume value of 1.6 ul/umole with the experimental data shown in Table I reveals that LUVETs composed of SPC and EPC exhibit trapped volumes smaller than those expected, while if a charged phospholipid species such as phosphatidylserine is present, the theoretical trapped volume is achieved.
- Two possible reasons for the low trapped volumes observed for EPC and SPC LUVETs are that there are a significant number of multilamellar vesicles present in the population, or that there are a greater proportion of small vesicles present than estimated from the freeze-fracture micrographs. The freeze-fracture results suggest that the number of multilamellar vesicles is very small (less than 2%), even if it is assumed that only 5% of fractured multilamellar systems exhibit a cross-fracture (see R. G. Miller, Nature, 287:166 (1980)). On the other hand, an underestimation of the number of small vesicles is likely.
- Moreover, as shown in Table I, the trapped volumes measured for EPC LUVs produced by the octylglucoside detergent dialysis procedure and the reverse phase evaporation procedure, which were subsequently extruded 10 times through a filter with a 100 nm pore size, are comparable to the trapped volumes obtained for the EPC LUVETs.
- These observations, taken together, demonstrate that the great majority of vesicles produced by the extrusion technique of the present invention are unilamellar, even though the measured trapped volume in certain cases is smaller than the calculated value.
- To establish that the procedures of the present invention when used with filters having a pore size of 100 nm produce LUVs, as opposed to SUVs, calorimetric studies were conducted on MLVs and LUVETs composed of 16:0/16:0 PC (dipalmitoylphosphatidylcholine-DPPC).
- SUVs composed of saturated phospholipids, such as, DPPC, are known to exhibit a reduction in the gel-liquid crystalline transition temperature (Te) and a broadening of the transition due to their highly curved membranes. This high curvature is generally considered undesirable because it results in increased disorder in the membrane's hydrocarbon region (see Schuh, J. R. Banerjee, U., Muller, L. and Chan. S. I. (1982) Biochim. Biophys. Acta, 687:219-225).
- In order to ascertain whether the LUVETs produced by the present invention are sufficiently large to avoid the problems arising from highly curved membranes, Te values were calorimetrically measured for MLVs and LUVETs prepared in accordance with the procedures of Example 1. The results are shown in Figure 5.
- As illustrated in this figure, the MLVs and LUVETs exhibit very similar values of Tc. These values are consistent with those reported in the literature. See Ladbrooke, B. D. and Chapman, D. (1969) Chem. Phys. Lipids, 3:304-367. They are in direct contrast to the behavior observed for sonicated DPPC vesicles, which exhibit a broadened gel-liquid crystalline transition which occurs some 4°C below the melting temperature of the multilamellar systems. See van Dijck, P. W. M., de Kruijff, B., Aarts, P. A. M. M., Verkleij, A. J. and de Gier, J. 1978) Biochim. Biophys. Acta, 506:183-191. Accordingly, the unilamellar liposomes prepared by the procedures of the present invention using filters with a 100 nm pore size are properly classified as LUVs, rather than SUVs.
- To test the structural integrity of the liposomes produced by the extrusion process of the present invention, LUVETs were prepared in accordance with the procedures of Example 1, but with a buffer having a NaCl concentration of 1 M, instead of 150 mM. After preparation, the liposomes were placed in distilled water creating a large osmotic pressure difference across the liposomes' membranes. Using arenazo III as a marker for liposome leakage, essentially no leakage was found under these severe test conditions.
- This example illustrates the use of a freeze-thaw procedure to increase the trapped volumes of the unilamellar liposomes produced by the present invention.
- SPC and EPC LUVETs prepared in accordance with the procedures of Example 1 were subjected to two freeze-thaw cycles (employing liquid nitrogen) followed by extrusion through new 100 nm pore size filters. Specifically, the LUVETs were placed in a plastic vial, and the vial placed in liquid nitrogen for approximately 1 minute. The frozen LUVETs were then thawed in water at room temperature for approximately 5 minutes. The thawed solution was extruded 3 times through new 100 nm filters, after which the freeze-thaw-extrude process was repeated a second time.
- Summary results for the process are given in Table I. Details of the size distribution of freeze-thawed SPC LUVETs is given in Figure 4 (solid columns).
- As shown in Figure 4, the mean diameter of the SPC LUVETs increased by approximately 20 nm. The calculated trapped volume for this vesicle distribution is 2.3 ul/umole which is in excellent agreement with the measured value of 2.2±0.1 ul/umol (Table I).
- Even higher trapped volumes were achieved using a soya PC system wherein freeze-thawing of LUVETs prepared by extrusion (10 times) through the 100 nm pore size filters, followed by extrusion (three to four times) through 200 nm pore size filters, resulted in trapped volumes on the order of 10 ul/umol phospholipid.
- An important parameter of LUV preparations is their trapping efficiency. This is especially so when the agents to be trapped are either expensive, as is the case for many drugs, or have low solubilities.
- In connection with the present invention, it has been found that the overall process can be made to have trapping efficiencies on the order of 30%, notwithstanding the relatively low trapped volumes of the vesicles produced, by simply increasing the lipid concentration of the solutions used to prepare the LUVETs.
- This effect is demonstrated in Figure 6 where the percentage of aqueous volume trapped inside the LUVETs is plotted against lipid concentration for LUVETs prepared in accordance with the procedures of Example 1 (solid circles) and using the freeze-thaw procedure of Example 4 (open circles).
- Preparation of LUVETs at lipid concentrations of 300 umoles/ml is easily accomplished, giving rise to trapping efficiencies on the order of 30% as shown in the figure. It is interesting to note that the freeze-thaw cycle only gives rise to significant increases in trapped volume per umol of lipid at lipid concentrations below 200 umol/ml. Similar observations have been reported by Pick, U. (1981) Arch. Biochem. Biophys., 212:186-194.
- This example illustrates the effects of using filters having pore sizes less than 100 nm on the size of the liposomes produced and on the number of passes through the filter needed to achieve substantial unilamellarity.
- MLVs were prepared in accordance with the procedures of Example 1 using egg PC at a concentration of 100 mg/ml and using a buffer of 150 mM NaCI and 20 mM Hepes (pH 7.5). The dispersion was passed ten times through two stacked polycarbonate filters having a pore size of either 50 nm or 30 nm using the apparatus of Figure 1A. Aliquots were taken after one and afterten passes through the extrusion device and used to prepare freeze-fracture micrographs as described above. Samples (4 ml, 25 mg phospholipid per ml) were also taken after various numbers of passes and analyzed by 31p NMR using Mn2+ as described above. The results are shown in Figures 7-9.
- As shown in Figure 7, vesicles extruded once through the 50 nm filters lost 37 percent of their 31p NMR signal upon addition of Mn2+, while vesicles extruded once through the 30 nm filters lost 47.5 percent. This indicates that the vesicles passed through the 50 nm filters are larger and/or more multilamellar than those passed through the 30 nm filter, a result which is confirmed by the freeze-fracture micrographs shown in Figures 8 and 9. Comparing the upper portions of those figures (Figures 8A and 9A) reveals that the liposomes which were passed once through the 50 nm filters are larger and more irregular than those which were passed once through the 30 nm filters.
- After ten passes, the 31P NMR signal intensities dropped by 53 and 56 percent for the 50 nm and 30 nm filters, respectively. This indicates that both filters were producing essentially the same size liposomes. This was confirmed by analysis of freeze-fracture micrographs which revealed that both populations had an average diameter of 44±14 nm, i.e., a diameter characteristic of SUVs. As illustrated by Figures 8B and 9B, in each case, the population produced was homogeneous.
- Comparing the curves of Figure 7 with the curve for the 100 nm filter in Figure 2 reveals that the 31P NMR signals tend to level off faster for the filters with smaller pore sizes. Accordingly, less passes through the extrusion apparatus are required to achieve a population of substantially unilamellar liposomes with the smaller pore size filters.
- This example illustrates the use of liposomes prepared in accordance with the present invention to deliver entrapped material in vivo. In particular, it illustrates for a rat model system the administration and subsequent in vivo distribution of 121|-tyraminyl-inulin (125|T|) containing LUVETs prepared in accordance with Example 1 above.
- Tyraminyl-inulin was prepared as follows. Inulin (1.0 g) was dissolved in 90.0 ml distilled H20 and cooled to 4°C, 10 ml (fresh) 0.1 M peridic acid was added and the solution was incubated at 4°C for 15 minutes in the dark. Periodate consumption was assayed by the arsenite method indicating approximately two oxidations per inulin molecule (see Dyer, J. in Methods of Biochemical Analysis, P. Glick (Ed.), Vol. 3, p. 111, Interscience (1956)). The reaction was terminated by neutralization with Ba(OH)2 and the periodate and iodate salts were removed by centrifugation. To the supernatant 4.3 g NaH2P04 and 0.55 g tyramine were added and the pH was adjusted to 7.5 with 1.0 M HCI. Subsequently, NaBH3CN (0.25 g) was added and the solution was stirred for 4 hr at room temperature. Remaining aldehyde groups were reduced by careful addition of 0.2 g NaBH4 and the solution was stirred for another hour at 27°C. Aliquots (25 ml) were degassed under reduced pressure and applied to a 1.5x80 cm Sephadex G-25 column previously equilibrated with H20 at 4°C. The flow rate was adjusted to 10 ml/hr and 4 ml fractions were collected. The fractions were assayed for tyramine by monitoring the absorbance at 279 nm and for sugar by employing the anthrone reagent technique (see Roe, J. H. (1955) J. Bio. Chem., 212:335-343). The sugar containing fraction eluted in the void volume and had a constant tyramine:inulin mole ratio of 0.6. The adduct was completely separated from the free tyramine and other salts as determined by rechromatography on Sephadex@ G-25. The peak fractions were lyophilized giving an 80% yield, based on inulin.
- The tyraminyl-inulin adduct was iodinated as follows. 2.5 mg of the tyraminyl-inulin adduct were dissolved in 0.2 ml HEPES (20 mM), NaCl (145 mM) pH 7.4 (HEPES buffered saline; HBS) and placed in a 1.5 ml stoppered vial in which 40 ug iodogen had been previously deposited from 300 uL CHCI3. Then carrier free Na1251 (4 mCi, 100 mCi/ml) was added and the reaction allowed to procede for 45 min at room temperature. The solution was then transferred to a vessel containing 10 ul 0.1 M Na2S20., 0.05 M KI which was then applied to a G-25 column (1 x20 cm) equilibrated with HBS. Fractions (0.5 ml) were collected and the 125| containing fractions eluting in the void volume (2.5 ml) were pooled. The resulting 125|-tyraminyl-inulin (125lTl) solution routinely contained 1 uCi/
uL 1251, where less than 0.01 % was in the free iodide form (less than 0.01 % was CHCI3 extractable when made to 1.2% H202 and 0.4% KI) and over 99% of the material eluted as one peak in the void volume on re-chromatography employing Sephadex G-25. In all studies the material was used within 2 weeks of production. - Liposomes loaded with 125|T| were prepared in accordance with the procedures described above. Specifically, 30 umol egg phosphatidylcholine (EPC) and 30 umol cholesterol were dried down from CHCI3. The resulting lipid film was dispersed in 1 ml HBS containing 1 mCi 125|T| by vortex mixing. The multilamellar systems thus produced were then extruded 10 times through two (stacked) polycarbonate Nuclepore filters (100 nm pore size) under N2 pressure (200-400 psi (1,378.94-2,757.88 kPa)). Aliquots (0.1 ml) of the LUVETs were applied to an Ultrogel Ac34 column (1 ml) previously equilibrated with HBS. The lipid containing fractions were pooled and rechromatography indicated that more than 97% of the 125|T| was "trapped" in the vesicles. The resulting liposome preparation had a trap volume of 0.9 ul/umol phospholipid as calculated from lipid phosphate analysis and entrapped 125lTl (see Fiske, C. H. and Subbarrow, Y. (1925) J. Biol. Chem., 66:375379). The average radius of these vesicles was 70 nm. The LUVETs containing 125|T| were diluted to 0.5 umol phospholipid in 200 ul of HBS, stored at 4°C and used within 2 days of preparation.
- The LUVET's were administered to female Wistar rats (150-200 g), which were fed ad libitum prior to and during the experiments, by lightly anesthetizing the animals with ether and then injecting 200 ul HBS containing approximately 0.5 uCi 125|T| encapsulated in LUVETs (0.5 umol phospholipid) via the tail vein. The rats were allowed to recover in metabolic cages where the urine and feces were collected. At various times post injection the rats were anesthetized with ether and sacrificed by exsanguination via the vena cava. Blood was collected in a syringe containing 200
uL 200 mM EDTA and recovery was approximately 85% assuming 4.9 ml blood/100 g rat. The heart, liver, lung, spleen and kidney were removed and the urine remaining in the bladder was collected. The carcass was then dissolved in 200 ml 9 M NaOH at 70°C overnight. Aliquots of carcass digest and samples of tissues were then assayed for the presence of 125|. - Figure 10 illustrates the clearance from the circulation of the LUVETs and the subsequent appearance of inulin in the urine. As shown in that figure, the encapsulated material in the circulation is initially rapidly reduced to approximately 40% of the injected levels, and thereafter decays with a much longer half-life (approximately 3 hr). Further, only 30% of the injected dose is eventually found in the urine even after 3 days. This latter result clearly indicates tissue uptake and retention of LUVET encapsulated 125|T|
- The actual tissue distributions are shown in Figure 11 where approximately 50% of the in vivo 1251T1 is accumulated by the liver, approximately 10% by the spleen and the rest is found in the carcass. Less than 3% 1251T1 was found in the heart, lung and kidney at any time post injection (data not shown).
- The tissue distributions observed are similar to those previously observed with liposomes produced by other methods (see, for example, Abra, R. M. and Hunt, C. A. (1981) Biochim. Biophys. Acta, 666493-503), thus demonstrating that the liposomes of the present invention are equivalent with regard to in vivo behavior to prior art liposomes.
- This example illustrates the production of liposomes directly from a lipid powder or pellet and buffer without the use of any solvents or other extraneous materials.
- One hundred mg of EPC, prepared as described above, was spooned into a test tube, 1.0 ml HEPES buffer was added, and the mixture was incubated at 20°C for 10 minutes. The mixture was briefly vortexed mixed for 2 minutes, followed by 5 minutes waiting time, followed by 2 minutes vortexing, and the resulting solution added to the pressure chamber of the apparatus of Figure 1A, which had been fitted with two stacked polycarbonate filters having a pore size of 100 nm. The solution was extruded through the filters ten times at a temperature of 20°C. The pressures employed were on the order of 200-300 psi (1,378.94-2,068.41 kPa), and the resulting flow rates were on the order of approximately 30 ml/min.
- Freeze fracutre micrographs of the resulting product were prepared following the procedures described above. The product was found to be a homogeneous population of substantially unilamellar liposomes having a mean diameter of approximately 70 nm as measured by freeze fracture. If desired, this mean diameter can be increased using the freeze-thaw procedures of Example 4 above.
- The procedures described above were repeated using 200 nm filters, instead of 100 nm filters. In this case, pressures on the order of 100 psi (689.47 kPa) were used, again resulting in flow rates of approximately 30 ml/min. Again, a homogeneous population of liposomes was produced, but in this case a substantial portion of the population was multilamellar, rather than unilamellar. The mean diameter of this population was approximately 168 nm, as measured by quasi-elastic light scattering using a
Nicomp Model 200 Laser Particle Sizer (Nicomp Instruments, Inc., Santa Barbara, California). - This example illustrates the preparation of a population of liposomes having a substantially unimodal distribution.
- Large multilamellar vesicles (MLVs) were prepared by the conventional process as follows. First, EPC prepared as described above was dissolved in chloroform and dried down and deposited as a film on the inside of a test tube. The MLVs were then formed by simply adding an aqueous buffer of 150 mM NaCI, 20 mM HEPES, pH 7.5, to the test tube and hydrating the lipid by vortex mixing.
- The resulting MLV dispersion was then transferred into the pressure chamber of the apparatus shown in Figure 1A, equipped with two stacked standard 25 mm polycarbonate filters having a pore size of 200 nm (Nuclepore, Inc., Pleasanton, California, Catalog # 110606). The dispersion was extruded through the
filters 25 times at a temperature of 20°C. The pressures employed were on the order of 100 psi (689.47 kPa), and the resulting flow rates were on the order of 30 ml/min. The sizing procedure was completed in less than approximately 15 minutes, and the resulting liposomes were found to be substantially intact, nothwithstanding their many passes through the filters. - The size distribution of the population at the end of the 25 passes was determined using the quasi-elastic light scattering technique described above. The results are shown in Table II, infra.
- As shown in the table, the population had a chi2 value of 1.42 indicating that a good fit was achieved by a second order polynomial, and thus that the diffusion coefficients of the vesicles had a unimodal Gaussian distribution. The mean diameter calculated for this population is 168 nm, i.e., about 15% smaller than the 200 nm pore size used for extrusion, and the standard deviation about the mean is a relatively narrow 55 nm.
- This example demonstrates the importance of using filters of a constant pore size, as opposed to a sequence of filters of decreasing pore sizes, to obtain a population of liposomes having a substantially unimodal size distribution.
- MLVs were prepared as in Example 9, but instead of being extruded 25 times through filters having the same pore size, they were extruded once through a series of filters having the following pore sizes: 1000 nm, 800 nm, 600 nm, 400 nm and 200 nm (Nuclepore, Inc., Pleasanton, California, Catalog Nos. 110610, 110609, 110608, 110607, and 110606). As in Example 9, the apparatus of Figure 1A was used, equipped in this case with just a single filter for each filter size. The pressures, flow rates and processing temperature were the same as in Example 9.
- The size distribution of the liposomes prepared in this manner, as determined by quasi-elastic light scattering, is shown in Table II. In this case, a huge chi2 value of 368 was calculated, which means that a second order polynomial did not fit the data, and thus that the diffusion coefficients of the liposomes do not have a unimodal Gaussian distribution.
- Comparing this result with the results for Example 9 clearly establishes that multiple passes of liposomes through filters of a constant pore size surprisingly produce a materially different size distribution from that produced by passage of the same type of liposomes through a series of filters of decreasing pore size.
- Although specific embodiments of the invention have been described and illustrated, it is to be understood that modifications can be made without departing from the invention's spirit and scope. For example, the invention can be practiced with a variety of membrane forming materials and encapsulatable solutes other than those illustrated in the examples. Similarly, various types of apparatus other than that illustrated herein can be used to practice the present invention. In particular, because each of its steps is easily controllable, the method of the present invention is especially suited for implementation in a totally automated manner, and such implementation is specifically included within the scope of the invention. Along these same lines, other types of equipment can be used to obtain autocorrelation functions, and other numerical approaches can be used to determine if the autocorrelation function is of the type that would be generated from a Gaussian distribution. The scope of the invention as defined in the appended claims is intended to cover these and other variations.
Claims (10)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT85903513T ATE55919T1 (en) | 1984-06-20 | 1985-06-19 | EXTRUSION PROCESS FOR GENERATION OF LIPOSOMES. |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62250284A | 1984-06-20 | 1984-06-20 | |
US62269084A | 1984-06-20 | 1984-06-20 | |
US622690 | 1984-06-20 | ||
US622502 | 1984-06-20 |
Publications (3)
Publication Number | Publication Date |
---|---|
EP0185756A1 EP0185756A1 (en) | 1986-07-02 |
EP0185756A4 EP0185756A4 (en) | 1987-09-02 |
EP0185756B1 true EP0185756B1 (en) | 1990-08-29 |
Family
ID=27089214
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP85903513A Expired - Lifetime EP0185756B1 (en) | 1984-06-20 | 1985-06-19 | Extrusion techniques for producing liposomes |
Country Status (11)
Country | Link |
---|---|
EP (1) | EP0185756B1 (en) |
JP (1) | JP2537186B2 (en) |
CA (1) | CA1264668A (en) |
DE (1) | DE3579426D1 (en) |
DK (1) | DK173397B1 (en) |
ES (1) | ES8608922A1 (en) |
GR (1) | GR851485B (en) |
IE (1) | IE58630B1 (en) |
PT (1) | PT80678B (en) |
SG (1) | SG21092G (en) |
WO (1) | WO1986000238A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19515163A1 (en) * | 1995-01-31 | 1996-08-01 | Erich Politsch | Reciprocating extruder and homogeniser for suspensions |
DE19542499A1 (en) * | 1995-11-15 | 1997-05-22 | Bayer Ag | Method and device for producing a parenteral drug preparation |
US9993427B2 (en) | 2013-03-14 | 2018-06-12 | Biorest Ltd. | Liposome formulation and manufacture |
Families Citing this family (60)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5231112A (en) * | 1984-04-12 | 1993-07-27 | The Liposome Company, Inc. | Compositions containing tris salt of cholesterol hemisuccinate and antifungal |
US5897873A (en) * | 1984-04-12 | 1999-04-27 | The Liposome Company, Inc. | Affinity associated vaccine |
US5736155A (en) * | 1984-08-08 | 1998-04-07 | The Liposome Company, Inc. | Encapsulation of antineoplastic agents in liposomes |
US5077056A (en) * | 1984-08-08 | 1991-12-31 | The Liposome Company, Inc. | Encapsulation of antineoplastic agents in liposomes |
US5059421A (en) * | 1985-07-26 | 1991-10-22 | The Liposome Company, Inc. | Preparation of targeted liposome systems of a defined size distribution |
MX9203291A (en) * | 1985-06-26 | 1992-08-01 | Liposome Co Inc | LIPOSOMAS COUPLING METHOD. |
US5041278A (en) * | 1985-10-15 | 1991-08-20 | The Liposome Company, Inc. | Alpha tocopherol-based vesicles |
US4737323A (en) * | 1986-02-13 | 1988-04-12 | Liposome Technology, Inc. | Liposome extrusion method |
US6759057B1 (en) | 1986-06-12 | 2004-07-06 | The Liposome Company, Inc. | Methods and compositions using liposome-encapsulated non-steroidal anti-inflammatory drugs |
US5252263A (en) * | 1986-06-16 | 1993-10-12 | The Liposome Company, Inc. | Induction of asymmetry in vesicles |
US6406713B1 (en) | 1987-03-05 | 2002-06-18 | The Liposome Company, Inc. | Methods of preparing low-toxicity drug-lipid complexes |
US5616334A (en) * | 1987-03-05 | 1997-04-01 | The Liposome Company, Inc. | Low toxicity drug-lipid systems |
MX9203808A (en) * | 1987-03-05 | 1992-07-01 | Liposome Co Inc | HIGH DRUG CONTENT FORMULATIONS: LIPID, FROM LIPOSOMIC-ANTINEOPLASTIC AGENTS. |
CA1338702C (en) * | 1987-03-05 | 1996-11-12 | Lawrence D. Mayer | High drug:lipid formulations of liposomal- antineoplastic agents |
JPH02502978A (en) * | 1987-04-16 | 1990-09-20 | ザ リポソーム カンパニー,インコーポレイテッド | Method and device for continuous size reduction of liposomes |
US5262168A (en) * | 1987-05-22 | 1993-11-16 | The Liposome Company, Inc. | Prostaglandin-lipid formulations |
US5082664A (en) * | 1987-05-22 | 1992-01-21 | The Liposome Company, Inc. | Prostaglandin-lipid formulations |
US5925375A (en) * | 1987-05-22 | 1999-07-20 | The Liposome Company, Inc. | Therapeutic use of multilamellar liposomal prostaglandin formulations |
MX9203804A (en) * | 1987-10-19 | 1992-07-01 | Liposome Co Inc | PHARMACEUTICAL SYSTEMS BASED ON TOCOPHEROL. |
US5948441A (en) * | 1988-03-07 | 1999-09-07 | The Liposome Company, Inc. | Method for size separation of particles |
DE3812816A1 (en) * | 1988-04-16 | 1989-11-02 | Lawaczeck Ruediger Dipl Phys P | METHOD FOR SOLUBILIZING LIPOSOMES AND / OR BIOLOGICAL MEMBRANES AND THE USE THEREOF |
EP0442962B1 (en) * | 1988-11-09 | 1994-01-05 | UNGER, Evan C | Liposomal radiologic contrast agents |
US5049392A (en) * | 1989-01-18 | 1991-09-17 | The Liposome Company, Inc. | Osmotically dependent vesicles |
ES2085920T3 (en) * | 1989-06-23 | 1996-06-16 | Liposome Co Inc | TARGETED LIPOSOMES AND LIPOSOMA-PROTEIN COUPLING METHODS. |
US5580575A (en) * | 1989-12-22 | 1996-12-03 | Imarx Pharmaceutical Corp. | Therapeutic drug delivery systems |
US5469854A (en) * | 1989-12-22 | 1995-11-28 | Imarx Pharmaceutical Corp. | Methods of preparing gas-filled liposomes |
US5352435A (en) * | 1989-12-22 | 1994-10-04 | Unger Evan C | Ionophore containing liposomes for ultrasound imaging |
US5149319A (en) * | 1990-09-11 | 1992-09-22 | Unger Evan C | Methods for providing localized therapeutic heat to biological tissues and fluids |
US5542935A (en) * | 1989-12-22 | 1996-08-06 | Imarx Pharmaceutical Corp. | Therapeutic delivery systems related applications |
US5820848A (en) * | 1990-01-12 | 1998-10-13 | The Liposome Company, Inc. | Methods of preparing interdigitation-fusion liposomes and gels which encapsulate a bioactive agent |
US5525232A (en) * | 1990-03-02 | 1996-06-11 | The Liposome Company, Inc. | Method for entrapment of cationic species in lemellar vesicles |
JP3533215B2 (en) * | 1990-07-31 | 2004-05-31 | ザ リポソーム カンパニー,インコーポレイテッド | Accumulation of amino acids and peptides in liposomes |
CA2091776C (en) * | 1990-10-05 | 1999-08-31 | Royden M. Coe | Liposome extrusion process |
US6623671B2 (en) | 1990-10-05 | 2003-09-23 | Royden M. Coe | Liposome extrusion process |
EP0804944A3 (en) | 1992-05-04 | 1998-08-26 | UNGER, Evan C | A method of providing a gas composition in a biological tissue or fluid in vivo |
CA2146963A1 (en) * | 1992-10-16 | 1994-04-28 | Andreas Sachse | Process and device for producing liquid, dispersed systems |
EP1118326A3 (en) * | 1993-05-21 | 2002-07-31 | The Liposome Company, Inc. | Reduction of liposome-induced adverse physiological reactions |
US5716526A (en) * | 1994-01-14 | 1998-02-10 | The Liposome Company, Inc. | Method of separating materials from liposomes or lipid complexes |
US6235308B1 (en) | 1994-06-10 | 2001-05-22 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Method of treating hypertension |
US5622715A (en) * | 1994-06-10 | 1997-04-22 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Method of improving renal function |
US5902604A (en) | 1995-06-06 | 1999-05-11 | Board Of Regents, The University Of Texas System | Submicron liposome suspensions obtained from preliposome lyophilizates |
NL1001380C2 (en) * | 1995-10-09 | 1997-04-11 | Fuji Photo Film Bv | Method of dispersing an oil droplet type emulsified material in a liquid supply system and coating method using such a dispersing method. |
DE69628909T2 (en) | 1995-10-12 | 2003-12-24 | Supergen Inc | LIPOSOME FORMULATION OF 5-BETA STEROIDS |
IT1289939B1 (en) * | 1997-02-20 | 1998-10-19 | Angelini Ricerche Spa | AQUEOUS PHARMACEUTICAL COMPOSITION INCLUDING A HIGHLY WATER INSOLUBLE ACTIVE SUBSTANCE |
US20010003580A1 (en) | 1998-01-14 | 2001-06-14 | Poh K. Hui | Preparation of a lipid blend and a phospholipid suspension containing the lipid blend |
JP2004528321A (en) | 2001-04-04 | 2004-09-16 | ノルディック ワクチン テクノロジー アクティーゼルスカブ | Polynucleotide binding complexes containing sterols and saponins |
PT1560641E (en) | 2002-11-14 | 2006-07-31 | Leuven K U Res & Dev | METHOD FOR THE PREPARATION OF EMULSES |
JP2005170923A (en) * | 2003-10-21 | 2005-06-30 | Konica Minolta Medical & Graphic Inc | Lyposome-containing x ray-imaging agent and method for producing the same |
JP2005170928A (en) * | 2003-10-21 | 2005-06-30 | Konica Minolta Medical & Graphic Inc | Lyposome-containing x ray-imaging agent and method for producing the same |
JP4654590B2 (en) * | 2004-03-31 | 2011-03-23 | コニカミノルタエムジー株式会社 | Contrast composition for X-ray CT and method for producing the same |
CA2650691C (en) | 2006-04-28 | 2015-10-06 | Children's Hospital Medical Center | Fusogenic properties of saposin c and related proteins and peptides for application to transmembrane drug delivery systems |
KR20080012605A (en) | 2006-08-04 | 2008-02-12 | 삼성에스디아이 주식회사 | Biomembrane devices with elastic energy barriers |
EP1920765A1 (en) * | 2006-11-07 | 2008-05-14 | Medigene AG | Liposome preparation by single-pass process |
US9445975B2 (en) * | 2008-10-03 | 2016-09-20 | Access Business Group International, Llc | Composition and method for preparing stable unilamellar liposomal suspension |
JP5771366B2 (en) | 2009-09-02 | 2015-08-26 | 株式会社バイオメッドコア | Liposome production apparatus and method |
CN103328038A (en) | 2010-12-01 | 2013-09-25 | 史拜诺莫度雷森公司 | Directed delivery of agents to neural anatomy |
JP2015509085A (en) | 2012-01-01 | 2015-03-26 | キュービーアイ エンタープライゼズ リミテッドQbi Enterprises Ltd. | Particles targeting ENDO180 for selective delivery of therapeutic and diagnostic agents |
ES2874305T3 (en) * | 2013-03-14 | 2021-11-04 | Zuli Holdings Ltd | Formulation and manufacture of liposomes |
US11246832B2 (en) * | 2016-06-28 | 2022-02-15 | Verily Life Sciences Llc | Serial filtration to generate small cholesterol-containing liposomes |
JP6403026B2 (en) * | 2017-06-29 | 2018-10-10 | バイオレスト リミテッド | Liposomes, formulations containing liposomes, and methods for producing formulations |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5186117A (en) * | 1975-01-27 | 1976-07-28 | Tanabe Seiyaku Co | Johoseibiryushiseizainoseiho |
US4263428A (en) * | 1978-03-24 | 1981-04-21 | The Regents Of The University Of California | Bis-anthracycline nucleic acid function inhibitors and improved method for administering the same |
CA1173360A (en) * | 1979-06-22 | 1984-08-28 | Jurg Schrank | Pharmaceutical preparations |
JPS5782310A (en) * | 1980-11-11 | 1982-05-22 | Tanabe Seiyaku Co Ltd | Production of liposome preparation |
-
1985
- 1985-06-07 CA CA000483485A patent/CA1264668A/en not_active Expired
- 1985-06-18 GR GR851485A patent/GR851485B/el unknown
- 1985-06-18 IE IE151485A patent/IE58630B1/en not_active IP Right Cessation
- 1985-06-19 WO PCT/US1985/001161 patent/WO1986000238A1/en active IP Right Grant
- 1985-06-19 EP EP85903513A patent/EP0185756B1/en not_active Expired - Lifetime
- 1985-06-19 JP JP60502838A patent/JP2537186B2/en not_active Expired - Lifetime
- 1985-06-19 DE DE8585903513T patent/DE3579426D1/en not_active Expired - Lifetime
- 1985-06-19 ES ES544364A patent/ES8608922A1/en not_active Expired
- 1985-06-20 PT PT80678A patent/PT80678B/en unknown
-
1986
- 1986-02-19 DK DK198600779A patent/DK173397B1/en not_active IP Right Cessation
-
1992
- 1992-03-04 SG SG210/92A patent/SG21092G/en unknown
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19515163A1 (en) * | 1995-01-31 | 1996-08-01 | Erich Politsch | Reciprocating extruder and homogeniser for suspensions |
DE19515163C2 (en) * | 1995-01-31 | 2002-11-28 | Erich Politsch | Device for extruding suspensions |
DE19542499A1 (en) * | 1995-11-15 | 1997-05-22 | Bayer Ag | Method and device for producing a parenteral drug preparation |
US9993427B2 (en) | 2013-03-14 | 2018-06-12 | Biorest Ltd. | Liposome formulation and manufacture |
US10265269B2 (en) | 2013-03-14 | 2019-04-23 | Biorest Ltd. | Liposome formulation and manufacture |
Also Published As
Publication number | Publication date |
---|---|
PT80678B (en) | 1987-08-19 |
EP0185756A4 (en) | 1987-09-02 |
JP2537186B2 (en) | 1996-09-25 |
ES544364A0 (en) | 1986-09-01 |
GR851485B (en) | 1985-11-25 |
SG21092G (en) | 1992-04-16 |
CA1264668C (en) | 1990-01-23 |
ES8608922A1 (en) | 1986-09-01 |
DK77986A (en) | 1986-02-19 |
PT80678A (en) | 1985-07-01 |
DK77986D0 (en) | 1986-02-19 |
WO1986000238A1 (en) | 1986-01-16 |
IE851514L (en) | 1985-12-20 |
CA1264668A (en) | 1990-01-23 |
JPS61502452A (en) | 1986-10-30 |
IE58630B1 (en) | 1993-10-20 |
DK173397B1 (en) | 2000-09-18 |
DE3579426D1 (en) | 1990-10-04 |
EP0185756A1 (en) | 1986-07-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0185756B1 (en) | Extrusion techniques for producing liposomes | |
US5008050A (en) | Extrusion technique for producing unilamellar vesicles | |
Mayer et al. | Vesicles of variable sizes produced by a rapid extrusion procedure | |
Pons et al. | Liposomes obtained by the ethanol injection method | |
Hope et al. | Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential | |
Hope et al. | Generation of multilamellar and unilamellar phospholipid vesicles | |
US5049392A (en) | Osmotically dependent vesicles | |
Zumbuehl et al. | Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid/detergent mixed micelles | |
US4994213A (en) | Method of preparing lipid structures | |
Boni et al. | Lipid-polyethylene glycol interactions: I. Induction of fusion between liposomes | |
US6447800B2 (en) | Method of loading preformed liposomes using ethanol | |
Hauser | Phospholipid vesicles | |
AU638245B2 (en) | Preparation of liposome and lipid complex compositions | |
Chapman et al. | Factors affecting solute entrapment in phospholipid vesicles prepared by the freeze-thaw extrusion method: a possible general method for improving the efficiency of entrapment | |
Elorza et al. | Comparison of particle size and encapsulation parameters of three liposomal preparations | |
Brekkan et al. | Properties of immobilized-liposome-chromatographic supports for interaction analysis | |
Vemuri et al. | Development and characterization of a liposome preparation by a pH-gradient method | |
Schenkman et al. | A kinetic and structural study of two-step aggregation and fusion of neutral phospholipid vesicles promoted by serum albumin at low pH | |
JP2537186C (en) | ||
Fresta et al. | Neutrase entrapment in stable multilamellar and large unilamellar vesicles for the acceleration of cheese ripening | |
Zakim et al. | [12] Spontaneous insertion of integral membrane proteins into preformed unilamellar vesicles | |
Beney et al. | Influence of the shape of phospholipid vesicles on the measurement of their size by photon correlation spectroscopy | |
Turanek | Fast-protein liquid chromatography system as a tool for liposome preparation by the extrusion procedure | |
Defrise-Quertain et al. | Spin label partitioning in lipid vesicles a model study for drug encapsulation | |
Katsai et al. | Preparation and characterization of liposomal delivery system of natural heme protein |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE FR GB IT LI LU NL SE |
|
17P | Request for examination filed |
Effective date: 19860602 |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 19870902 |
|
17Q | First examination report despatched |
Effective date: 19880722 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AT BE CH DE FR GB IT LI LU NL SE |
|
REF | Corresponds to: |
Ref document number: 55919 Country of ref document: AT Date of ref document: 19900915 Kind code of ref document: T |
|
REF | Corresponds to: |
Ref document number: 3579426 Country of ref document: DE Date of ref document: 19901004 |
|
ET | Fr: translation filed | ||
ITF | It: translation for a ep patent filed |
Owner name: CON LOR S.R.L. |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
ITTA | It: last paid annual fee | ||
26N | No opposition filed | ||
K2C2 | Correction of patent specification (partial reprint) published |
Effective date: 19900829 |
|
EPTA | Lu: last paid annual fee | ||
EAL | Se: european patent in force in sweden |
Ref document number: 85903513.1 |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: IF02 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: NL Payment date: 20040528 Year of fee payment: 20 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: AT Payment date: 20040603 Year of fee payment: 20 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20040616 Year of fee payment: 20 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20040618 Year of fee payment: 20 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: SE Payment date: 20040621 Year of fee payment: 20 Ref country code: CH Payment date: 20040621 Year of fee payment: 20 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: LU Payment date: 20040701 Year of fee payment: 20 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: BE Payment date: 20040715 Year of fee payment: 20 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: DE Payment date: 20040802 Year of fee payment: 20 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GB Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION Effective date: 20050618 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NL Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION Effective date: 20050619 |
|
BE20 | Be: patent expired |
Owner name: THE *LIPOSOME CY INC. Effective date: 20050619 |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: PE20 |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
NLV7 | Nl: ceased due to reaching the maximum lifetime of a patent |
Effective date: 20050619 |
|
EUG | Se: european patent has lapsed | ||
REG | Reference to a national code |
Ref country code: GB Ref legal event code: 732E |
|
BE20 | Be: patent expired |
Owner name: THE *LIPOSOME CY INC. Effective date: 20050619 |